1. |
- Bergdahl, Andreas, et al.
(author)
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Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TRPC1.
- 2003
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In: Circulation Research. - 0009-7330. ; 93:9, s. 839-847
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Journal article (peer-reviewed)abstract
- The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-ß-cyclodextrin (mßcd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ETA receptor) was reduced by mßcd and increased by cholesterol. Neither ligand binding nor colocalization of ETA and caveolin-1 was affected by mßcd. Ca2+ inflow via store-operated channels after depletion of intracellular Ca2+ stores was reduced in mßcd-treated caudal arteries, as shown by Mn2+ quench rate and intracellular [Ca2+] response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase–polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by mßcd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni2+ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1–induced contractions were insensitive to the TRPC1 blocking antibody and to mßcd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to mßcd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1.
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2. |
- Dreja, Karl, et al.
(author)
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Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction.
- 2002
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In: Arteriosclerosis, Thrombosis and Vascular Biology. - 1524-4636. ; 22:8, s. 1267-1272
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Journal article (peer-reviewed)abstract
- OBJECTIVE: This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility. Methods and Results- Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-beta-cyclodextrin, and the effects on force and Ca2+ handling were evaluated. In cholesterol-depleted preparations, the force responses to alpha1-adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 micro mol/L AlCl3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by >50%. The rise in global intracellular free Ca2+ concentration in response to 5-HT was attenuated, as was the generation of Ca2+ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT2A receptors, but not alpha1-adrenergic receptors, in the caveolin-1-containing fractions, suggesting localization of the former to caveolae. CONCLUSIONS: These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae.
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3. |
- Gomez-Pinilla, Pedro J., et al.
(author)
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Effect of melatonin on age associated changes in guinea pig bladder function
- 2007
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In: Journal of Urology. - : Ovid Technologies (Wolters Kluwer Health). - 1527-3792 .- 0022-5347. ; 177:4, s. 1558-1561
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Journal article (peer-reviewed)abstract
- Purpose: The incidence of urinary incontinence increases with age but the cause and effect relationship between aging and altered bladder function is poorly understood. It was suggested that melatonin can ameliorate negative effects induced by aging by its free radical scavenging activity and its ability to decrease oxidative stress. We investigated the changes in bladder function evoked by aging and the possible benefits of melatonin treatment on age related bladder disturbances. Materials and Methods: Bladder function was assessed using cystometry in conscious, freely moving female guinea pigs. Animals were grouped according to age as young adults (4 months old) and senescents (18 to 20 months old). A group of senescent animals were treated with 2.5 mg kg(-1) day(-1) melatonin for 21 days. Results: Aging led to increased detrusor activity, as demonstrated by short micturition intervals, decreased bladder capacity and spontaneous contractions during the filling phase. During the voiding phase aged animals showed lower micturition pressures than young adults. Melatonin counteracted the cystometric changes in senescent animals and restored micturition parameters to those of young adults. Conclusions: These results show that in guinea pigs aging induces detrusor overactivity. Melatonin treatment improved age induced changes in bladder function. If similar effects can be demonstrated in humans, melatonin treatment may be a new approach to decrease the impact of age related bladder disorders.
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4. |
- Gomez-Pinilla, Pedro J, et al.
(author)
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Melatonin restores impaired contractility in aged guinea pig urinary bladder
- 2008
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In: Journal of Pineal Research. - : Blackwell Publishing Ltd. - 1600-079X .- 0742-3098. ; 44:4, s. 416-425
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Journal article (peer-reviewed)abstract
- Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin-treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca2+](i)) was estimated by epifluorescence microscopy of fura-2-loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca2+](i) in response to the stimuli, suggesting a reduced Ca(2+)sensitivity. Indeed, the agonist-induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca2+ sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI-17 and MYPT1, and with the absence of CPI-17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca2+ handling and Ca2+ sensitizations pathways. Moreover, the indoleamine restored age-induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging-related urinary bladder contractile impairment.
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5. |
- Lindqvist, Anders, et al.
(author)
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Effects of oxygen tension on energetics of cultured vascular smooth muscle.
- 2002
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In: American Journal of Physiology: Heart and Circulatory Physiology. - : American Physiological Society. - 1522-1539 .- 0363-6135. ; 283:1, s. 110-117
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Journal article (peer-reviewed)abstract
- Chronic hypoxia is a clinically important condition known to cause vascular abnormalities. To investigate the cellular mechanisms involved, we kept rings of a rat tail artery for 4 days in hypoxic culture (HC) or normoxic culture (NC) (PO(2) = 14 vs. 110 mmHg) and then measured contractility, oxygen consumption (JO(2)), and lactate production (J(lac)) in oxygenated medium. Compared with fresh rings, basal ATP turnover (J(ATP)) was decreased in HC, but not in NC, with a shift from oxidative toward glycolytic metabolism. JO(2) during mitochondrial uncoupling was reduced by HC but not by NC. Glycogen stores were increased 40-fold by HC and fourfold by NC. Maximum tension in response to norepinephrine and the JO(2) versus tension relationship (JO(2) vs. high K(+) elicited force) were unaffected by either HC or NC. Force transients in response to caffeine were increased in HC, whereas intracellular Ca(2+) wave activity during adrenergic stimulation was decreased. Protein synthesis rate was reduced by HC. The results show that long-term hypoxia depresses basal energy turnover, impairs mitochondrial capacity, and alters Ca(2+) homeostasis, but does not affect contractile energetics. These alterations may form a basis for vascular damage by chronic hypoxia.
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6. |
- Malmqvist, Ulf, et al.
(author)
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Female pig urethral tone is dependent on Rho guanosine triphosphatases and Rho-associated kinase.
- 2004
-
In: Journal of Urology. - : Ovid Technologies (Wolters Kluwer Health). - 1527-3792 .- 0022-5347. ; 171:5, s. 1955-1958
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Journal article (peer-reviewed)abstract
- PURPOSE: Circular smooth muscle of the urethra generates spontaneous myogenic tone of relevance for the maintenance of continence. We tested if Rho guanosine triphosphatases (GTPases) and Rho-associated kinase (ROK) are involved in the generation of urethral tone. MATERIALS AND METHODS: Small circular strips of female pig urethra were dissected out and mounted for recording isometric force. The effect of pharmacological agents known to modulate the activity of Rho GTPases or ROK was examined. The intracellular calcium concentration was measured using fura-2. RESULTS: Urethral tone was abolished by removing extracellular calcium or by adding the calcium antagonist felodipine. The decrease in force was closely related to a decrease in intracellular calcium concentration, indicating that tone depends on membrane associated mechanisms. Toxin B, which inactivates Rho GTPases, and Y 27632, which inhibits ROK, completely abolished tone in the female pig urethra. The latter effect occurred without any change in the intracellular calcium concentration. CONCLUSIONS: The results suggests that urethral tone depends on activity in G-protein coupled pathways and inhibition of this activity is sufficient for urethral tone relaxation. Thus, to our knowledge a new pathway in the generation of urethral tone, which might be acted on by autonomic nerves during micturition, has been identified.
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7. |
- Swärd, Karl, et al.
(author)
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Contractile effects of polycations in permeabilized smooth muscle
- 1998
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In: Journal of Muscle Research and Cell Motility. - 0142-4319. ; 19:5, s. 463-472
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Journal article (peer-reviewed)abstract
- The polycations spermine, neomycin and polylysine potentiated Ca(2+)-activated force in beta-escin permeabilized guinea-pig ileum strips. The effect was inhibited by the calmodulin antagonists trifluoperazine, mastoparan and W13. Potentiation was slow or absent in chi-toxin permeabilized strips, indicating dependence on penetration of the polycations into cells. The effects of spermine and neomycin were maintained after extensive permeabilization by beta-escin, which eliminated the contractile effect of GTPgammaS. Replacement of ATP by CTP, which is not a substrate for myosin light chain kinase, inhibited contractile potentiation. Potentiation of Ca(2+)-activated contractions was associated with increased phosphorylation of the myosin regulatory light chains (LC20). A contractile effect of polylysine and neomycin was also seen in Ca(2+)-free medium and after partial LC20 thiophosphorylation, indicating that phosphorylation-independent processes may contribute to the response. Although spermine does not cause contraction in Ca(2+)-free medium at physiological [MgATP], it did so when [MgATP] was lowered to 40 micron. Similar to high-[Mg2+], the rate of contraction on addition of ATP to strips incubated with microcystin-LR in inhibit phosphatase activity was increased by the polycations, but only at [Ca2+] < 0.3 micron. The results suggest that polycations increase Ca(2+)-activated force by inhibiting myosin phosphatase activity, thereby increasing myosin LC20 phosphorylation. However, additional activation mechanisms, evident at low [Ca2+] and at low [ATP] and possibly involving direct activation of myosin, contribute to their effect.
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8. |
- Swärd, Karl, et al.
(author)
-
Influence of mitochondrial inhibition on global and local [Ca(2+)](I) in rat tail artery.
- 2002
-
In: Circulation Research. - 0009-7330. ; 90:7, s. 792-799
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Journal article (peer-reviewed)abstract
- Inhibition of oxidative metabolism is often found to decrease contractility of systemic vascular smooth muscle, but not to reduce global [Ca(2+)](i). In the present study, we probe the hypothesis that it is associated with an altered pattern of intracellular Ca(2+) oscillations (waves) influencing force development. In the rat tail artery, mitochondrial inhibitors (rotenone, antimycin A, and cyanide) reduced alpha(1)-adrenoceptor-stimulated force by 50% to 80%, but did not reduce global [Ca(2+)](i). Less relaxation (about 30%) was observed after inhibition of myosin phosphatase activity with calyculin A, suggesting that part of the metabolic sensitivity involves the regulation of myosin 20-kDa light chain phosphorylation, although no decrease in phosphorylation was found in freeze-clamped tissue. Confocal imaging revealed that the mitochondrial inhibitors increased the frequency but reduced the amplitude of asynchronous cellular Ca(2+) waves elicited by alpha(1) stimulation. The altered wave pattern, in association with increased basal [Ca(2+)](i), accounted for the unchanged global [Ca(2+)](i). Inhibition of glycolytic ATP production by arsenate caused similar effects on Ca(2+) waves and global [Ca(2+)](i), developing gradually in parallel with decreased contractility. Inhibition of wave activity by the InsP(3) receptor antagonist 2-APB correlated closely with relaxation. Furthermore, abolition of waves with thapsigargin in the presence of verapamil reduced force by about 50%, despite unaltered global [Ca(2+)](i), suggesting that contraction may at least partly depend on Ca(2+) wave activity. This study therefore indicates that mitochondrial inhibition influences Ca(2+) wave activity, possibly due to a close spatial relationship of mitochondria and the sarcoplasmic reticulum and that this contributes to metabolic vascular relaxation.
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9. |
- Swärd, Karl, et al.
(author)
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Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum
- 2000
-
In: Journal of Physiology. - 1469-7793. ; 522, s. 33-49
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Journal article (peer-reviewed)abstract
- Ca2+ sensitization of smooth muscle contraction involves the small GTPase RhoA, inhibition of myosin light chain phosphatase (MLCP) and enhanced myosin regulatory light chain (LC20) phosphorylation. A potential effector of RhoA is Rho-associated kinase (ROK). The role of ROK in Ca2+ sensitization was investigated in guinea-pig ileum. Contraction of permeabilized muscle strips induced by GTPgammaS at pCa 6.5 was inhibited by the kinase inhibitors Y-27632, HA1077 and H-7 with IC50 values that correlated with the known Ki values for inhibition of ROK. GTPgammaS also increased LC20 phosphorylation and this was prevented by HA1077. Contraction and LC20 phosphorylation elicited at pCa 5.75 were, however, unaffected by HA1077. Pre-treatment of intact tissue strips with HA1077 abolished the tonic component of carbachol-induced contraction and the sustained elevation of LC20 phosphorylation, but had no effect on the transient or sustained increase in [Ca2+]i induced by carbachol. LC20 phosphorylation and contraction dynamics suggest that the ROK-mediated increase in LC20 phosphorylation is due to MLCP inhibition, not myosin light chain kinase activation. In the absence of Ca2+, GTPgammaS stimulated 35S incorporation from [35S]ATPgammaS into the myosin targeting subunit of MLCP (MYPT). The enhanced thiophosphorylation was inhibited by HA1077. No thiophosphorylation of LC20 was detected. These results indicate that ROK mediates agonist-induced increases in myosin phosphorylation and force by inhibiting MLCP activity through phosphorylation of MYPT. Under Ca2+-free conditions, ROK does not appear to phosphorylate LC20 in situ, in contrast to its ability to phosphorylate myosin in vitro. In particular, ROK activation is essential for the tonic phase of agonist-induced contraction.
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10. |
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11. |
- Alajbegovic, Azra, et al.
(author)
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MRTFA overexpression promotes conversion of human coronary artery smooth muscle cells into lipid-laden foam cells
- 2021
-
In: Vascular Pharmacology. - : Elsevier BV. - 1537-1891. ; 138
-
Journal article (peer-reviewed)abstract
- Objective: Smooth muscle cells contribute significantly to lipid-laden foam cells in atherosclerotic plaques. However, the underlying mechanisms transforming smooth muscle cells into foam cells are poorly understood. The purpose of this study was to gain insight into the molecular mechanisms regulating smooth muscle foam cell formation. Approach and results: Using human coronary artery smooth muscle cells we found that the transcriptional co-activator MRTFA promotes lipid accumulation via several mechanisms, including direct transcriptional control of LDL receptor, enhanced fluid-phase pinocytosis and reduced lipid efflux. Inhibition of MRTF activity with CCG1423 and CCG203971 significantly reduced lipid accumulation. Furthermore, we demonstrate enhanced MRTFA expression in vascular remodeling of human vessels. Conclusions: This study demonstrates a novel role for MRTFA as an important regulator of lipid homeostasis in vascular smooth muscle cells. Thus, MRTFA could potentially be a new therapeutic target for inhibition of vascular lipid accumulation.
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12. |
- Alajbegovic, Azra, et al.
(author)
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Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization
- 2017
-
In: Biochimica et Biophysica Acta - Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1864:6, s. 1088-1098
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Journal article (peer-reviewed)abstract
- The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA.In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells. This analysis was performed using cardiovascular disease-focused miRNA arrays in both mouse and human cells. The potential clinical importance of actin polymerization in aortic aneurysm was evaluated using biopsies from mildly dilated human thoracic aorta in patients with stenotic tricuspid or bicuspid aortic valve.By integrating information from multiple qPCR based miRNA arrays we identified a group of five miRNAs (miR-1, miR-22, miR-143, miR-145 and miR-378a) that were sensitive to actin polymerization and MRTF-A overexpression in both mouse and human vascular smooth muscle. With the exception of miR-22, these miRNAs were also relatively enriched in striated and/or smooth muscle containing tissues. Actin polymerization was found to be dramatically reduced in the aorta from patients with mild aortic dilations. This was associated with a decrease in actin/MRTF-regulated miRNAs.In conclusion, the transcriptional co-activator MRTF-A and actin polymerization regulated a subset of miRNAs in vascular smooth muscle. Identification of novel miRNAs regulated by actin/MRTF-A may provide further insight into the mechanisms underlying vascular disease states, such as aortic aneurysm, as well as novel ideas regarding therapeutic strategies. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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13. |
- Albinsson, Sebastian, et al.
(author)
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Arterial remodeling and plasma volume expansion in caveolin-1 deficient mice.
- 2007
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In: American Journal of Physiology: Regulatory, Integrative and Comparative Physiology. - : American Physiological Society. - 0363-6119 .- 1522-1490. ; 293, s. 1222-1231
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Journal article (peer-reviewed)abstract
- Caveolin- 1 ( Cav- 1) is essential for the morphology of membrane caveolae and exerts a negative influence on a number of signaling systems, including nitric oxide ( NO) production and activity of the MAP kinase cascade. In the vascular system, ablation of caveolin- 1 may thus be expected to cause arterial dilatation and increased vessel wall mass ( remodeling). This was tested in Cav- 1 knockout ( KO) mice by a detailed morphometric and functional analysis of mesenteric resistance arteries, shown to lack caveolae. Quantitative morphometry revealed increased media thickness and media- to- lumen ratio in KO. Pressure- induced myogenic tone and flow- induced dilatation were decreased in KO arteries, but both were increased toward wild- type ( WT) levels following NO synthase ( NOS) inhibition. Isometric force recordings following NOS inhibition showed rightward shifts of passive and active length- force relationships in KO, and the force response to alpha 1- adrenergic stimulation was increased. In contrast, media thickness and force response of the aorta were unaltered in KO vs. WT, whereas lumen diameter was increased. Mean arterial blood pressure during isoflurane anesthesia was not different in KO vs. WT, but greater fluctuation in blood pressure over time was noted. Following NOS inhibition, fluctuations disappeared and pressure increased twice as much in KO ( 38 +/- 6%) compared with WT ( 17 +/- 3%). Tracer- dilution experiments showed increased plasma volume in KO. We conclude that NO affects blood pressure more in Cav- 1 KO than in WT mice and that restructuring of resistance vessels and an increased responsiveness to adrenergic stimulation compensate for a decreased tone in Cav- 1 KO mice.
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14. |
- Albinsson, Sebastian, et al.
(author)
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Differential dependence of stretch and shear stress signaling on caveolin-1 in the vascular wall
- 2008
-
In: American Journal of Physiology: Cell Physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 294, s. 271-279
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Journal article (peer-reviewed)abstract
- The role of caveolae in stretch- vs. flow-induced vascular responses was investigated using caveolin-1 deficient (KO) mice. Portal veins were stretched longitudinally for 5 min (acute) or 72 h (organ culture). Basal ERK1/2 and Akt phosphorylation were increased in organ-cultured KO veins, as were protein synthesis and vessel wall cross-section. Stretch stimulated acute phosphorylation of ERK1/2 and long-term phosphorylation of focal adhesion kinase (FAK) and cofilin, but did not affect Akt phosphorylation. Protein synthesis, and particularly synthesis of smooth muscle differentiation markers, was increased by stretch. These effects did not differ in portal veins from KO and control mice, which also showed the same contractile response to membrane depolarization and inhibition by the Rho kinase inhibitor Y-27632. KO carotid arteries had increased wall cross-section and responded to pressurization (120 mmHg) for 1 h with increased ERK1/2 but not Akt phosphorylation, similar to control arteries. Shear stress by flow for 15 min, on the other hand, increased phosphorylation of Akt in carotids from control but not KO mice. In conclusion, caveolin-1 contributes to a low basal ERK1/2 and Akt activity and is required for Akt-dependent signals in response to shear stress (flow), but is not essential for trophic effects of stretch (pressure) in the vascular wall. Key words: Hypertrophy, vasoconstriction, vascular smooth muscle, endothelium, nitric oxide.
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15. |
- Albinsson, Sebastian, et al.
(author)
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Targeting smooth muscle microRNAs for therapeutic benefit in vascular disease.
- 2013
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In: Pharmacological Research. - : Elsevier BV. - 1096-1186 .- 1043-6618. ; 75:April,22, s. 28-36
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Research review (peer-reviewed)abstract
- In view of the bioinformatic projection that a third of all protein coding genes and essentially all biological pathways are under control of microRNAs (miRNAs), it is not surprising that this class of small RNAs plays roles in vascular disease progression. MiRNAs have been shown to be involved in cholesterol turnover, thrombosis, glucose homeostasis and vascular function. Some miRNAs appear to be specific for certain cells, and the role that such cell-specific miRNAs play in vascular disease is only beginning to be appreciated. A notable example is the miR-143/145 cluster which is enriched in mature and highly differentiated smooth muscle cells (SMCs). Here we outline and discuss the recent literature on SMC-expressed miRNAs in major vascular diseases, including atherosclerosis, neointima formation, aortic aneurysm formation, and pulmonary arterial hypertension. Forced expression of miR-145 emerges as a promising strategy for reduction and stabilization of atherosclerotic plaques as well as for reducing neointimal hyperplasia. It is concluded that if obstacles in the form of delivery and untoward effects of antimirs and mimics can be overcome, the outlook for targeting of SMC-specific miRNAs for therapeutic benefit in vascular disease is bright.
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16. |
- Andersson, Kristina E, et al.
(author)
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Diverse effects of oats on cholesterol metabolism in C57BL/6 mice correlate with expression of hepatic bile acid-producing enzymes.
- 2013
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In: European Journal of Nutrition. - : Springer Science and Business Media LLC. - 1436-6215 .- 1436-6207. ; 52:7, s. 1755-1769
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Journal article (peer-reviewed)abstract
- PURPOSE: We previously reported that two substrains of C57BL/6 mice respond differently to oats with respect to reduction in plasma cholesterol. Analysis of this difference might offer clues to mechanisms behind the cholesterol-lowering effect of oats. Here, we address the possible roles of hepatic steroid metabolism and the intestinal microbiota in this respect. METHODS: Female C57BL/6 mice were fed an atherogenic diet with oat bran (27 %) or control fibres for 4 weeks. RESULTS: C57BL/6 NCrl mice responded to oat bran with 19 ± 1 % (P < 0.001) lower plasma cholesterol, 40 ± 5 % (P < 0.01) higher excretion of bile acids and increased expression of the bile acid-producing hepatic enzymes CYP7A1 and CYP8B1, but none of these effects were found in C57BL/6JBomTac mice. However, on control diet, C57BL/6JBomTac had tenfold higher expression of CYP7A1 and levels of hepatic cholesterol esters than C57BL/6NCrl mice. Plasma levels of fructosamine indicated improved glycemic control by oat bran in C57BL/6NCrl but not in C57BL/6JBomTac. C57BL/6JBomTac had higher intestinal microbiota diversity, but lower numbers of Enterobacteriaceae, Akkermansia and Bacteroides Fragilis than C57BL/6NCrl mice. Oat bran increased bacterial numbers in both substrains. Microbiota diversity was reduced by oats in C57BL/6JBomTac, but unaffected in C57BL/6NCrl. CONCLUSIONS: Our data do not support a connection between altered microbiota diversity and reduced plasma cholesterol, but the bacterial composition in the intestine may influence the effects of added fibres. The cholesterol-lowering properties of oats involve increased production of bile acids via the classical pathway with up-regulation of CYP7A1 and CYP8B1. Altered cholesterol or bile acid metabolism may interfere with the potential of oats to reduce plasma cholesterol.
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17. |
- Andersson, Kristina E, et al.
(author)
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Effects of oats on plasma cholesterol and lipoproteins in C57BL/6 mice are substrain specific.
- 2010
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In: British Journal of Nutrition. - 1475-2662. ; 103, s. 513-521
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Journal article (peer-reviewed)abstract
- Cholesterol-lowering effects of oats have been demonstrated in both animals and human subjects. However, the crucial properties of oat-containing diets that determine their health effects need to be further investigated to optimise their use. A mouse model would be a valuable tool, but few such studies have been published to date. We investigated the effects of oat bran on plasma cholesterol and lipoproteins in two substrains of C57BL/6 mice. Western diet was made atherogenic by the addition of 0.8 % cholesterol and 0.1 % cholic acid. After 4 weeks on atherogenic diet, total plasma cholesterol had increased from 1.86-2.53 to 3.77-4.40 mmol/l. In C57BL/6NCrl mice, inclusion of 27 and 40 % oat bran reduced total plasma cholesterol by 19 and 24 %, respectively, reduced the shift from HDL to LDL+VLDL and caused increased faecal cholesterol excretion. There was no effect of oat bran on plasma levels of the inflammatory markers fibrinogen, serum amyloid A or TNF-alpha. Contrary to findings in C57BL/6NCrl mice, there was no sustained effect of oat bran (27 or 40 %) on plasma cholesterol in C57BL/6JBomTac mice after 4 weeks of feeding. Thus, C57BL/6NCrl mice fed an atherogenic diet are a good model for studies of physiological effects of oats, whereas a substrain derived from C57BL/6J, raised in a different breeding environment and likely possessing functional genetic differences from C57BL/6N, is considerably less responsive to oats. The present finding that two substrains of mice respond differently to oats is of practical value, but can also help to elucidate mechanisms of the cholesterol-lowering effect of oats.
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18. |
- Andersson, Linda, 1973, et al.
(author)
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Glucosylceramide synthase deficiency in the heart compromises β1-adrenergic receptor trafficking
- 2021
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In: European Heart Journal. - : Oxford University Press. - 0195-668X .- 1522-9645. ; 42:43, s. 4481-4492
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Journal article (peer-reviewed)abstract
- AIMS: Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function.METHODS AND RESULTS: Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg-/- mice). In 9- to 10-week-old hUgcg-/- mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg-/- mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to β-adrenergic stimulation was reduced in cardiomyocytes from hUgcg-/- mice and that Ugcg knockdown suppressed the internalization and trafficking of β1-adrenergic receptors.CONCLUSIONS: Our findings suggest that cardiac glycosphingolipids are required to maintain β-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.
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19. |
- Arévalo-Martínez, Marycarmen, et al.
(author)
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Myocardin-Dependent Kv1.5 Channel Expression Prevents Phenotypic Modulation of Human Vessels in Organ Culture
- 2019
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In: Arteriosclerosis, Thrombosis, and Vascular Biology. - 1524-4636. ; 39:12, s. 273-286
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Journal article (peer-reviewed)abstract
- OBJECTIVE: We have previously described that changes in the expression of Kv channels associate to phenotypic modulation (PM), so that Kv1.3/Kv1.5 ratio is a landmark of vascular smooth muscle cells phenotype. Moreover, we demonstrated that the Kv1.3 functional expression is relevant for PM in several types of vascular lesions. Here, we explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels, and the mechanisms linking the switch in Kv1.3 /Kv1.5 ratio to PM. Approach and Results: Vascular remodeling was explored using organ culture and primary cultures of vascular smooth muscle cells obtained from human vessels. We studied the effects of Kv1.3 inhibition on serum-induced remodeling, as well as the impact of viral vector-mediated overexpression of Kv channels or myocardin knock-down. Kv1.3 blockade prevented remodeling by inhibiting proliferation, migration, and extracellular matrix secretion. PM activated Kv1.3 via downregulation of Kv1.5. Hence, both Kv1.3 blockers and Kv1.5 overexpression inhibited remodeling in a nonadditive fashion. Finally, myocardin knock-down induced vessel remodeling and Kv1.5 downregulation and myocardin overexpression increased Kv1.5, while Kv1.5 overexpression inhibited PM without changing myocardin expression. CONCLUSIONS: We demonstrate that Kv1.5 channel gene is a myocardin-regulated, vascular smooth muscle cells contractile marker. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed in dedifferentiated cells. We demonstrated that the inhibition of Kv1.3 channel function with selective blockers or by preventing Kv1.5 downregulation can represent an effective, novel strategy for the prevention of intimal hyperplasia and restenosis of the human vessels used for coronary angioplasty procedures.
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20. |
- Bankell, Elisabeth, et al.
(author)
-
Suppression of smooth muscle cell inflammation by myocardin-related transcription factors involves inactivation of TANK-binding kinase 1
- 2024
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In: Scientific Reports. - 2045-2322. ; 14:1
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Journal article (peer-reviewed)abstract
- Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in human smooth muscle cells (SMCs) through sequestration of RelA in the NF-κB complex, but additional mechanisms are likely involved. The cGAS-STING pathway is activated by double-stranded DNA in the cytosolic compartment and acts through TANK-binding kinase 1 (TBK1) to spark inflammation. The present study tested if MRTFs suppress inflammation also by targeting cGAS-STING signaling. Interrogation of a transcriptomic dataset where myocardin was overexpressed using a panel of 56 cGAS-STING cytokines showed the panel to be repressed. Moreover, MYOCD, MRTFA, and SRF associated negatively with the panel in human arteries. RT-qPCR in human bronchial SMCs showed that all MRTFs reduced pro-inflammatory cytokines on the panel. MRTFs diminished phosphorylation of TBK1, while STING phosphorylation was marginally affected. The TBK1 inhibitor amlexanox, but not the STING inhibitor H-151, reduced the anti-inflammatory effect of MRTF-A. Co-immunoprecipitation and proximity ligation assays supported binding between MRTF-A and TBK1 in SMCs. MRTFs thus appear to suppress cellular inflammation in part by acting on the kinase TBK1. This may defend SMCs against pro-inflammatory insults in disease.
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21. |
- Bankell, Elisabeth, et al.
(author)
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The antimicrobial peptide LL-37 triggers release of apoptosis-inducing factor and shows direct effects on mitochondria
- 2022
-
In: Biochemistry and Biophysics Reports. - : Elsevier BV. - 2405-5808. ; 29
-
Journal article (peer-reviewed)abstract
- The human antimicrobial peptide LL-37 permeabilizes the plasma membrane of host cells, but LL-37-induced direct effects on mitochondrial membrane permeability and function has not been reported. Here, we demonstrate that LL-37 is rapidly (within 20 min) internalized by human osteoblast-like MG63 cells, and that the peptide co-localizes with MitoTracker arguing for accumulation in mitochondria. Subcellular fractionation and Western blot disclose that stimulation with LL-37 (8 μM) for 2 h triggers release of the mitochondrial protein apoptosis-inducing factor (AIF) to the cytosol, whereas LL-37 causes no release of cytochrome C oxidase subunit IV of the inner mitochondrial membrane, suggesting that LL-37 affects mitochondrial membrane permeability in a specific manner. Next, we investigated release of AIF and cytochrome C from isolated mitochondria by measuring immunoreactivity by dot blot. The media of mitochondria treated with LL-37 (8 μM) for 2 h contained 50% more AIF and three times more cytochrome C than that of control mitochondria, showing that LL-37 promotes release of both AIF and cytochrome C. Moreover, in vesicles reflecting mitochondrial membrane lipid composition, LL-37 stimulates membrane permeabilization and release of tracer molecules. We conclude that LL-37 is rapidly internalized by MG63 cells and accumulates in mitochondria, and that the peptide triggers release of pro-apoptotic AIF and directly affects mitochondrial membrane structural properties.
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22. |
- Bergdahl, Andreas, et al.
(author)
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Caveolae-associated signalling in smooth muscle
- 2004
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In: Canadian Journal of Physiology and Pharmacology. - : Canadian Science Publishing. - 0008-4212 .- 1205-7541. ; 82:5, s. 289-299
-
Research review (peer-reviewed)abstract
- Caveolae are flask-shaped invaginations in the membrane that depend on the contents of cholesterol and on the structural protein caveolin. The organisation of caveolae in parallel strands between dense bands in smooth muscle is arguably unique. It is increasingly recognised, bolstered in large part by recent studies in caveolae deficient animals, that caveolae sequester and regulate a variety of signalling intermediaries. The role of caveolae in smooth muscle signal transduction, as inferred from studies on transgenic animals and in vitro approaches, is the topic of the current review. Both G-protein coupled receptors and tyrosine kinase receptors are believed to cluster in caveolae, and the exciting possibility that caveolae provide a platform for interactions between the sarcoplasmic reticulum and plasmalemmal ion channels is emerging. Moreover, messengers involved in Ca2+ sensitization of myosin phosphorylation and contraction may depend on caveolae or caveolin. Caveolae thus appear to constitute an important signalling domain that plays a role not only in regulation of smooth muscle tone, but also in proliferation, such as seen in neointima formation and atherosclerosis.
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23. |
- Bergdahl, Andreas, et al.
(author)
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Lovastatin Induces Relaxation and Inhibits L-Type Ca2+ Current in the Rat Basilar Artery.
- 2003
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In: Pharmacology and Toxicology. - : Wiley. - 1600-0773 .- 0901-9928. ; 93:3, s. 128-134
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Journal article (peer-reviewed)abstract
- Statins inhibit cholesterol biosynthesis and protect against ischaemic stroke. It has become increasingly apparent that the beneficial effects of statin therapy may extend beyond lowering of serum cholesterol. The present study was done to explore possible pleiotropic statin effects at the level of the cerebral vascular smooth muscle. Lovastatin, lovastatin acid, simvastatin and pravastatin, were added to segments of the rat basilar artery and effects on contraction and Ca2+ handling were examined. Pravastatin had no effect on contraction. Simvastatin, lovastatin, and, to a lesser degree, lovastatin acid, caused relaxation (IC50=0.8, 1.9 and 22 μmol/l) of both intact and denuded arteries precontracted with 5-HT or high-K+. This effect was not reversed by mevalonate, suggesting that it was not related to cholesterol or isoprenoid metabolism. Relaxation was associated with a reduction of the intracellular Ca2+ concentration measured with Fura 2 and with a reduced Mn2+ quench rate, suggesting a direct effect on ion channels in the smooth muscle cell membrane. Current measurements in isolated and voltage clamped basilar artery muscle cells demonstrated that both lovastatin and lovastatin acid inhibit L-type Ca2+ current. We propose that lipophilicity is an important factor behind the effects of statins on vascular tone and that Ca2+ current inhibition is the likely mechanism of action.
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24. |
- Bhattachariya, Anirban, et al.
(author)
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Expression of microRNAs is essential for arterial myogenic tone and pressure-induced activation of the PI3-kinase/Akt pathway.
- 2014
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In: Cardiovascular Research. - : Oxford University Press (OUP). - 1755-3245 .- 0008-6363. ; 101:2, s. 288-296
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Journal article (peer-reviewed)abstract
- The myogenic response is the intrinsic ability of small arteries to constrict in response to increased intraluminal pressure. Although microRNAs have been shown to play a role in vascular smooth muscle function, their importance in the regulation of the myogenic response is not known. In this study, we investigate the role of microRNAs in the regulation of myogenic tone by using smooth muscle-specific and tamoxifen-inducible deletion of the endonuclease Dicer in mice.
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25. |
- Bhattachariya, Anirban, et al.
(author)
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Spontaneous activity and stretch-induced contractile differentiation are reduced in vascular smooth muscle of miR-143/145 knockout mice.
- 2015
-
In: Acta Physiologica. - : Wiley. - 1748-1716 .- 1748-1708. ; 215:3, s. 133-143
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Journal article (peer-reviewed)abstract
- Stretch is essential for maintaining the contractile phenotype of vascular smooth muscle cells, and small non-coding microRNAs are known to be important in this process. Using a Dicer knockout model, we have previously reported that microRNAs are essential for stretch-induced differentiation and regulation of L-type calcium channel expression. The aim of this study was to investigate the importance of the smooth muscle-enriched miR-143/145 microRNA cluster for stretch-induced differentiation of the portal vein.
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26. |
- Cavalera, Michele, et al.
(author)
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Dietary rose hip exerts antiatherosclerotic effects and increases nitric oxide-mediated dilation in ApoE-null mice
- 2017
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In: Journal of Nutritional Biochemistry. - : Elsevier BV. - 0955-2863. ; 44, s. 52-59
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Journal article (peer-reviewed)abstract
- Atherosclerosis is a disease in which atheromatous plaques develop inside arteries, leading to reduced or obstructed blood flow that in turn may cause stroke and heart attack. Rose hip is the fruit of plants of the genus Rosa, belonging to the Rosaceae family, and it is rich in antioxidants with high amounts of ascorbic acid and phenolic compounds. Several studies have shown that fruits, seeds and roots of these plants exert antidiabetic, antiobesity and cholesterol-lowering effects in rodents as well as humans. The aim of this study was to elucidate the mechanisms by which rose hip lowers plasma cholesterol and to evaluate its effects on atherosclerotic plaque formation. ApoE-null mice were fed either an HFD (CTR) or HFD with rose hip supplementation (RH) for 24 weeks. At the end of the study, we found that blood pressure and atherosclerotic plaques, together with oxidized LDL, total cholesterol and fibrinogen levels were markedly reduced in the RH group. Fecal cholesterol content, liver expression of Ldlr and selected reverse cholesterol transport (RCT) genes such as Abca1, Abcg1 and Scarb1 were significantly increased upon RH feeding. In the aorta, the scavenger receptor Cd36 and the proinflammatory Il1β genes were markedly down-regulated compared to the CTR mice. Finally, we found that RH increased nitric oxide-mediated dilation of the caudal artery. Taken together, these results suggest that rose hip is a suitable dietary supplement for preventing atherosclerotic plaques formation by modulating systemic blood pressure and the expression of RCT and inflammatory genes.
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27. |
- Dahan, Diana, et al.
(author)
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Induction of angiotensin converting enzyme after miR-143/145 deletion is critical for impaired smooth muscle contractility.
- 2014
-
In: American Journal of Physiology: Cell Physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 307:12, s. 1093-1101
-
Journal article (peer-reviewed)abstract
- MicroRNAs have emerged as regulators of smooth muscle cell phenotype with a role in smooth muscle-related disease. Studies have shown that miR-143 and miR-145 are the most highly expressed microRNAs in smooth muscle cells, controlling differentiation and function. The effect of miR-143/145 knockout has been established in the vasculature but not in smooth muscle from other organs. Using knockout mice we found that maximal contraction induced by either depolarization or phosphatase inhibition was reduced in vascular and airway smooth muscle but maintained in the urinary bladder. Furthermore, a reduction of media thickness and reduced expression of differentiation markers was seen in the aorta but not in the bladder. Supporting the view that phenotype switching depends on a tissue-specific target of miR-143/145, we found induction of angiotensin converting enzyme in the aorta but not in the bladder where angiotensin converting enzyme was expressed at a low level. Chronic treatment with angiotensin type-1 receptor antagonist restored contractility in miR-143/145-deficient aorta while leaving bladder contractility unaffected. This shows that tissue-specific targets are critical for the effects of miR-143/145 on smooth muscle differentiation and that angiotensin converting enzyme is one such target.
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28. |
- Dahan, Diana, et al.
(author)
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MicroRNA-Dependent Control of Serotonin-Induced Pulmonary Arterial Contraction
- 2017
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In: Journal of Vascular Research. - : S. Karger AG. - 1018-1172 .- 1423-0135. ; 54:4, s. 246-256
-
Journal article (peer-reviewed)abstract
- Background: Serotonin (5-HT) is considered to play a role in pulmonary arterial hypertension by regulating vascular remodeling and smooth muscle contractility. Here, arteries from mice with inducible and smooth muscle-specific deletion of Dicer were used to address mechanisms by which microRNAs control 5-HT-induced contraction. Methods: Mice were used 5 weeks after Dicer deletion, and pulmonary artery contractility was analyzed by wire myography. Results: No change was seen in right ventricular systolic pressure following dicer deletion, but systemic blood pressure was reduced. Enhanced 5-HT-induced contraction in Dicer KO pulmonary arteries was associated with increased 5-HT2A receptor mRNA expression whereas 5-HT1B and 5-HT2B receptor mRNAs were unchanged. Contraction by the 5-HT2A agonist TCB-2 was increased in Dicer KO as was the response to the 5-HT2B agonist BW723C86. Effects of Src and protein kinase C inhibition were similar in control and KO arteries, but the effect of inhibition of Rho kinase was reduced. We identified miR-30c as a potential candidate for 5-HT2A receptor regulation as it repressed 5-HT2A mRNA and protein. Conclusion: Our findings show that 5-HT receptor signaling in the arterial wall is subject to regulation by microRNAs and that this entails altered 5-HT2A receptor expression and signaling.
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29. |
- Dahl, Sara, et al.
(author)
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Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells
- 2020
-
In: Inflammation Research. - : Springer Science and Business Media LLC. - 1420-908X .- 1023-3830. ; 69:6, s. 579-588
-
Journal article (peer-reviewed)abstract
- OBJECTIVE: The importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA) signaling in human vascular smooth muscle cells.MATERIALS AND METHODS: Cellular import of LL-37 and synthetic dsRNA (poly I:C) were investigated by immunocytochemistry and fluorescence imaging. Transcript and protein expression were determined by qPCR, ELISA and Western blot. Knockdown of TLR3 was performed by siRNA.RESULTS: LL-37 was rapidly internalized, suggesting that it has intracellular actions. Co-stimulation with poly I:C and LL-37 enhanced pro-inflammatory IL-6 and MCP-1 transcripts several fold compared to treatment with poly I:C or LL-37 alone. Poly I:C increased IL-6 and MCP-1 protein production, and this effect was potentiated by LL-37. LL-37-induced stimulation of poly I:C signaling was not associated with enhanced import of poly I:C. Treatment with poly I:C and LL-37 in combination increased expression of dsRNA receptor TLR3 compared to stimulation with poly I:C or LL-37 alone. In TLR3 knockdown cells, treatment with poly I:C and LL-37 in combination had no effect on IL-6 and MCP-1 expression, showing loss of function.CONCLUSIONS: LL-37 potentiates dsRNA-induced cytokine production through up-regulation of TLR3 expression representing a novel pro-inflammatory mechanism.
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30. |
- Daoud, Fatima, et al.
(author)
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Inducible Deletion of YAP and TAZ in Adult Mouse Smooth Muscle Causes Rapid and Lethal Colonic Pseudo-Obstruction
- 2021
-
In: Cellular and Molecular Gastroenterology and Hepatology. - : Elsevier BV. - 2352-345X. ; 11:2, s. 623-637
-
Journal article (peer-reviewed)abstract
- Background & AimsYAP (Yap1) and TAZ (Wwtr1) are transcriptional co-activators and downstream effectors of the Hippo pathway, which play crucial roles in organ size control and cancer pathogenesis. Genetic deletion of YAP/TAZ has shown their critical importance for embryonic development of the heart, vasculature, and gastrointestinal mesenchyme. The aim of this study was to determine the functional role of YAP/TAZ in adult smooth muscle cells in vivo.MethodsBecause YAP and TAZ are mutually redundant, we used YAP/TAZ double-floxed mice crossed with mice that express tamoxifen-inducible CreERT2 recombinase driven by the smooth muscle–specific myosin heavy chain promoter.ResultsDouble-knockout of YAP/TAZ in adult smooth muscle causes lethality within 2 weeks, mainly owing to colonic pseudo-obstruction, characterized by severe distension and fecal impaction. RNA sequencing in colon and urinary bladder showed that smooth muscle markers and muscarinic receptors were down-regulated in the YAP/TAZ knockout. The same transcripts also correlated with YAP/TAZ in the human colon. Myograph experiments showed reduced contractility to depolarization by potassium chloride and a nearly abolished muscarinic contraction and spontaneous activity in colon rings of YAP/TAZ knockout.ConclusionsYAP and TAZ in smooth muscle are guardians of colonic contractility and control expression of contractile proteins and muscarinic receptors. The knockout model has features of human chronic intestinal pseudo-obstruction and may be useful for studying this disease.
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31. |
- Daoud, Fatima, et al.
(author)
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Role of smooth muscle YAP and TAZ in protection against phenotypic modulation, inflammation, and aneurysm development
- 2022
-
In: Biochemical Pharmacology. - : Elsevier BV. - 0006-2952. ; 206
-
Research review (peer-reviewed)abstract
- A ruptured arterial aneurysm, especially in the aorta, represents one of the most acute and mortal conditions encountered in clinical medicine. Population-based screening in elderly men, treatment of risk factors, such as hypertension, and endovascular or open repair of rupture-prone lesions, represent cornerstones in management. Surgical repair has a sizeable effect on life-expectancy, but medical therapy that retards aneurysm growth still represents a considerable and unmet clinical need. In the current review we survey recent findings implicating the mechano-responsive transcriptional co-activators YAP and TAZ in protection from aneurysm development. Arteries from mouse mutants that lack YAP and TAZ in vascular smooth muscle respond inadequately to mechanical stimulation, and they develop aneurysms characterized by elastin fragmentation, proteoglycan infiltration, and severe inflammation at breathtaking speed. This seems to be due, at least in part, to unscheduled activation of STING (stimulator of interferon genes), an arm of innate immunity that responds to double-stranded DNA in the cytoplasm. YAP and TAZ protect from STING activation by securing nuclear integrity. These novel insights suggest unanticipated medical therapies for sporadic and genetic aneurysms alike, involving inhibition of kinases in the Hippo pathway using small molecules, or inhibition of STING signaling itself. Translation of these novel findings into clinical therapies now represents an important priority.
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32. |
- Daoud, Fatima, et al.
(author)
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YAP and TAZ in Vascular Smooth Muscle Confer Protection Against Hypertensive Vasculopathy
- 2022
-
In: Arteriosclerosis, Thrombosis, and Vascular Biology. - 1079-5642. ; 42:4, s. 428-443
-
Journal article (peer-reviewed)abstract
- Background: Hypertension remains a major risk factor for cardiovascular diseases, but the underlying mechanisms are not well understood. We hypothesize that appropriate mechanotransduction and contractile function in vascular smooth muscle cells are crucial to maintain vascular wall integrity. The Hippo pathway effectors YAP (yes-associated protein 1) and TAZ (WW domain containing transcription regulator 1) have been identified as mechanosensitive transcriptional coactivators. However, their role in vascular smooth muscle cell mechanotransduction has not been investigated in vivo. Methods: We performed physiological and molecular analyses utilizing an inducible smooth muscle-specific YAP/TAZ knockout mouse model. Results: Arteries lacking YAP/TAZ have reduced agonist-mediated contraction, decreased myogenic response, and attenuated stretch-induced transcriptional regulation of smooth muscle markers. Moreover, in established hypertension, YAP/TAZ knockout results in severe vascular lesions in small mesenteric arteries characterized by neointimal hyperplasia, elastin degradation, and adventitial thickening. Conclusions: This study demonstrates a protective role of YAP/TAZ against hypertensive vasculopathy.
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33. |
- De Luca, Francesco, 1976, et al.
(author)
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Identification of ARMH4 and WIPF3 as human podocyte proteins with potential roles in immunomodulation and cytoskeletal dynamics
- 2023
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 18:1
-
Journal article (peer-reviewed)abstract
- The podocyte is a specialized cell type critically involved in maintaining the selective filtration barrier of the kidney. Podocytes are primary or secondary targets for a multitude of kidney diseases. Despite intense investigation, the transcriptome and proteome of human podocytes remain incompletely characterized. Here, we analyzed publicly available RNA-Seq data from human kidneys (n = 85) to computationally identify potential novel podocyte markers. For confirmation, we used an online histology resource followed by in-house staining of human kidneys and biochemical fractionation of glomeruli. Initial characterization of the novel podocyte transcripts was performed using viral overexpression and mRNA silencing. Several previously unrecognized gene products were identified that correlated to established podocyte markers on the RNA level and that were histologically localized to podocytes. ARMH4 (a.k.a. UT2 or C14orf37) and WIPF3 (a.k.a CR16) were among the hits. We show that these transcripts increase in response to overexpression of the podocyte transcription factor LMX1B. Overexpression of ARMH4 from low endogenous levels in primary kidney epithelial cells reduced the release of the inflammatory mediators IL-1B and IL-8 (CXCL8). The opposite effect was seen in mature human podocytes when ARMH4 was silenced. Overexpression of WIPF3 stabilized N-WASP, known to be required for maintenance of podocyte foot processes, and increased cell motility as shown using a scratch assay. Moreover, data from normal and diseased human kidneys showed that ARMH4 was downregulated in glomerular pathologies, while WIPF3 remained constantly expressed. ARMH4 and WIPF3 are new potential markers of human podocytes, where they may modulate inflammatory insults by controlling cytokine release and contribute to cytoskeletal dynamics, respectively.
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34. |
- De Santis, Martina M, et al.
(author)
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Extracellular-Matrix-Reinforced Bioinks for 3D Bioprinting Human Tissue
- 2021
-
In: Advanced Materials. - : Wiley. - 1521-4095 .- 0935-9648. ; 33:3
-
Journal article (peer-reviewed)abstract
- Recent advances in 3D bioprinting allow for generating intricate structures with dimensions relevant for human tissue, but suitable bioinks for producing translationally relevant tissue with complex geometries remain unidentified. Here, a tissue-specific hybrid bioink is described, composed of a natural polymer, alginate, reinforced with extracellular matrix derived from decellularized tissue (rECM). rECM has rheological and gelation properties beneficial for 3D bioprinting while retaining biologically inductive properties supporting tissue maturation ex vivo and in vivo. These bioinks are shear thinning, resist cell sedimentation, improve viability of multiple cell types, and enhance mechanical stability in hydrogels derived from them. 3D printed constructs generated from rECM bioinks suppress the foreign body response, are pro-angiogenic and support recipient-derived de novo blood vessel formation across the entire graft thickness in a murine model of transplant immunosuppression. Their proof-of-principle for generating human tissue is demonstrated by 3D bioprinting human airways composed of regionally specified primary human airway epithelial progenitor and smooth muscle cells. Airway lumens remained patent with viable cells for one month in vitro with evidence of differentiation into mature epithelial cell types found in native human airways. rECM bioinks are a promising new approach for generating functional human tissue using 3D bioprinting.
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35. |
- Ekman, Mari, et al.
(author)
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Association of muscarinic M(3) receptors and Kir6.1 with caveolae in human detrusor muscle.
- 2012
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In: European Journal of Pharmacology. - : Elsevier BV. - 1879-0712 .- 0014-2999. ; 683:1-3, s. 238-245
-
Journal article (peer-reviewed)abstract
- Caveolae are 50-100nm large membrane invaginations that play a role in cellular signaling. The aim of the present study was to assess whether muscarinic M(3) receptors and the K(ATP) channel subunit Kir6.1 are associated with human detrusor caveolae, and to pharmacologically assess the relevance of this organization for contractility. Detrusor strips were dissected and used in ultrastructural, biochemical and mechanical studies. Caveolae were manipulated by cholesterol desorption using mβcd (methyl-β-cyclodextrin). Mβcd disrupted caveolae and caused a cholesterol-dependent ~3-fold rightward shift of the concentration-response curve for the muscarinic receptor agonist carbachol. The effect of mβcd was inhibited by the K(ATP) blockers glibenclamide, repaglinide and PNU-37883, and it was mimicked by the K(ATP) activator levcromakalim. Immunoelectron microscopy showed muscarinic M(3) receptors and Kir6.1 to be enriched in caveolae. In conclusion, pharmacological K(ATP) channel inhibition antagonizes the effect of caveolae disruption on muscarinic contractility in the human detrusor, and the K(ATP) channel subunit Kir6.1 co-localizes with M(3) receptors in caveolae.
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36. |
- Ekman, Mari, et al.
(author)
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HIF-mediated metabolic switching in bladder outlet obstruction mitigates the relaxing effect of mitochondrial inhibition.
- 2014
-
In: Laboratory Investigation. - : Elsevier BV. - 1530-0307 .- 0023-6837. ; 94:5, s. 557-568
-
Journal article (peer-reviewed)abstract
- Prior work demonstrated increased levels of hypoxia-inducible factor-1α (HIF-1α) in the bladder following outlet obstruction, associated with bladder growth and fibrosis. Here we hypothesized that HIF induction in outlet obstruction also switches energetic support of contraction from mitochondrial respiration to glycolysis. To address this hypothesis, we created infravesical outlet obstruction in female Sprague-Dawley rats and examined HIF induction and transcriptional activation. HIF-1α increased after 6 weeks of outlet obstruction as assessed by western blotting and yet transcription factor-binding site analysis indicated HIF activation already at 10 days of obstruction. Accumulation HIF-2α and of Arnt2 proteins were found at 10 days, providing an explanation for the lack of correlation between HIF-1α protein and transcriptional activation. HIF signature targets, including Slc2a1, Tpi1, Eno1 and Ldha increased in obstructed compared with sham-operated bladders. The autophagy markers Bnip3 and LC3B-II were also increased at 6 week of obstruction, but electron microscopy did not support mitophagy. Mitochondria were, however, remodeled with increased expression of Cox4 compared with other markers. In keeping with a switch toward glycolytic support of contraction, we found that relaxation by the mitochondrial inhibitor cyanide was reduced in obstructed bladders. This was mimicked by organ culture with the HIF-inducer dimethyloxalylglycine, which also upregulated expression of Ldha. On the basis of these findings, we conclude that HIF activation in outlet obstruction involves mechanisms beyond the accumulation of HIF-1α protein and that it results in a switch of the energetic support of contraction to anaerobic glycolysis. This metabolic adaptation encompasses increased expression of glucose transporters and glycolytic enzymes combined with mitochondrial remodeling. Together, these changes uphold contractility when mitochondrial respiration is limited.Laboratory Investigation advance online publication, 3 March 2014; doi:10.1038/labinvest.2014.48.
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37. |
- Ekman, Mari, et al.
(author)
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MicroRNAs in Bladder Outlet Obstruction: Relationship to Growth and Matrix Remodelling.
- 2016
-
In: Basic & Clinical Pharmacology & Toxicology. - : Wiley. - 1742-7843 .- 1742-7835. ; 119:S3, s. 5-17
-
Research review (peer-reviewed)abstract
- The discovery of microRNAs (miRNAs), which are ~22 nucleotide RNAs that inhibit protein synthesis in a sequence-specific manner and are present in a range of species, has born hope of new therapeutic strategies. miRNAs play important roles in development "Development" of what??and disease, but they remain poorly studied in uropathologies beyond cancer. Here, we discuss biological functions of miRNAs in the lower urogenital tract. A special focus is on miRNAs that change in bladder outlet obstruction (BOO). This is a condition that affects nearly one third of all men over 60 years and that involves growth and fibrosis of the urinary bladder. Animal models of BOO, such as that in rat, have been developed and they feature a massive 6-fold bladder growth over 6 weeks. Using microarrays, we have charted the miRNAs that change during the time-course of this process and identified several with important modulatory roles. We discuss known and predicted functions of miR-1, miR-29, miR-30, miR-132/212, miR-204 and miR-221, all of which change in BOO. The majority of the miRNA-mediated influences in BOO are expected to favour growth. We also outline evidence that miR-29 represents a key effector molecule in a generic response to mechanical distension that is designed to counteract exaggerated organ deformation via effects on matrix deposition and stiffness. We conclude that miRNAs play important roles in bladder remodelling and growth and that they may be targeted pharmacologically to combat diseases of the lower urinary tract. This article is protected by copyright. All rights reserved.
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38. |
- Ekman, Mari, et al.
(author)
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Mir-29 repression in bladder outlet obstruction contributes to matrix remodeling and altered stiffness.
- 2013
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:12
-
Journal article (peer-reviewed)abstract
- Recent work has uncovered a role of the microRNA (miRNA) miR-29 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-29 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMAD3, all of which repress miR-29, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMAD3 phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMAD3 activation, respectively, correlated with repression of miR-29b and miR-29c at 10 days of obstruction and with repression of miR-29c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-29 following outlet obstruction was associated with increased levels of miR-29 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-29 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-29c and miR-29c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-29 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMAD3 reduce miR-29 in bladder outlet obstruction, and where the resulting drop in miR-29 contributes to matrix remodeling and altered passive mechanical properties of the detrusor.
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39. |
- Ekman, Mari, et al.
(author)
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Neurite outgrowth in cultured mouse pelvic ganglia - Effects of neurotrophins and bladder tissue
- 2017
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In: Autonomic Neuroscience: Basic & Clinical. - : Elsevier BV. - 1566-0702. ; 205, s. 41-49
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Journal article (peer-reviewed)abstract
- Neurotrophic factors regulate survival and growth of neurons. The urinary bladder is innervated via both sympathetic and parasympathetic neurons located in the major pelvic ganglion. The aim of the present study was to characterize the effects of the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on the sprouting rate of sympathetic and parasympathetic neurites from the female mouse ganglion. The pelvic ganglion was dissected out and attached to a petri dish and cultured in vitro. All three factors (BDNF, NT-3 and NGF) stimulated neurite outgrowth of both sympathetic and parasympathetic neurites although BDNF and NT-3 had a higher stimulatory effect on parasympathetic ganglion cells. The neurotrophin receptors TrkA, TrkB and TrkC were all expressed in neurons of the ganglia. Co-culture of ganglia with urinary bladder tissue, but not diaphragm tissue, increased the sprouting rate of neurites. Active forms of BDNF and NT-3 were detected in urinary bladder tissue using western blotting whereas tissue from the diaphragm expressed NGF. Neurite outgrowth from the pelvic ganglion was inhibited by a TrkB receptor antagonist. We therefore suggest that the urinary bladder releases trophic factors, including BDNF and NT-3, which regulate neurite outgrowth via activation of neuronal Trk-receptors. These findings could influence future strategies for developing pharmaceuticals to improve re-innervation due to bladder pathologies.
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40. |
- Gil-Ramírez, Alicia, et al.
(author)
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Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes
- 2020
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10
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Journal article (peer-reviewed)abstract
- Vascular bio-scaffolds produced from decellularized tissue offer a promising material for treatment of several types of cardiovascular diseases. These materials have the potential to maintain the functional properties of the extracellular matrix (ECM), and allow for growth and remodeling in vivo. The most commonly used methods for decellularization are based on chemicals and enzymes combinations, which often damage the ECM and cause cytotoxic effects in vivo. Mild methods involving pressurized CO2-ethanol (EtOH)-based fluids, in a supercritical or near supercritical state, have been studied for decellularization of cardiovascular tissue, but results are controversial. Moreover, data are lacking on the amount and type of lipids remaining in the tissue. Here we show that pressurized CO2-EtOH-H2O fluids (average molar composition, ΧCO2 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized CO2-limonene fluids (ΧCO2 0.88) and neat supercritical CO2 (scCO2) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized CO2-limonene followed by enzyme treatment, resulted in efficient DNA removal. The structure of elastic fibers was preserved after pressurized treatment, regardless solvent composition. In conclusion, pressurized CO2-ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO2-limonene fluids facilitate subsequent enzymatic removal of DNA.
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41. |
- Gomez, Maria, et al.
(author)
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Long-term regulation of contractility and calcium current in smooth muscle
- 1997
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In: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 273:5, s. 1714-1720
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Journal article (peer-reviewed)abstract
- Longitudinal smooth muscle strips from guinea pig ileum were cultured in vitro for 5 days, and the relationship between extracellular Ca2+ and force in high-K+ medium was evaluated. In strips cultured with 10% fetal calf serum (FCS), this relationship was shifted to the right (50% effective concentration changed by 2-3 mM) compared with strips cultured without FCS. The shift was prevented by inclusion of verapamil (1 microM) during culture and mimicked by ionomycin in the absence of FCS. The intracellular Ca2+ concentration ([Ca2+]i) during stimulation with high-K+ solution or carbachol was reduced after culture with FCS, whereas the [Ca2+]i-force relationship was unaffected. Cells were isolated from cultured strips, and whole cell voltage-clamp experiments were performed. Maximum inward Ca2+ current (10 mM Ba2+), normalized to cell capacitance, was almost three times smaller in cells isolated from strips cultured with FCS. Culture with 1 microM verapamil prevented this reduction. These results suggest that increased [Ca2+]i during culture downregulates Ca2+ current density, with associated effects on contractility.
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42. |
- Grossi, Mario, et al.
(author)
-
Inhibition of Polyamine Formation Antagonizes Vascular Smooth Muscle Cell Proliferation and Preserves the Contractile Phenotype.
- 2014
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In: Basic & Clinical Pharmacology & Toxicology. - : Wiley. - 1742-7843 .- 1742-7835. ; 115:5, s. 379-388
-
Journal article (peer-reviewed)abstract
- The polyamines putrescine, spermidine and spermine play essential roles in cell proliferation and migration, two processes involved in the development of vascular disease. Thus, intervention with polyamine formation may represent a way to inhibit unwanted vascular smooth muscle cell (VSMC) proliferation. The aim of the present study was to assess the importance of polyamines for VSMC proliferation and vascular contractility. The rate-limiting step in polyamine biosynthesis is catalyzed by ornithine decarboxylase. Treatment with α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, reduced DNA synthesis in primary rat VSMCs in a concentration-dependent manner with an IC50 value of 100 μM. Moreover, DFMO reduced VSMC migration assessed in a scratch assay. The DFMO-induced attenuation of VSMC proliferation was associated with lowered cellular amount of polyamines. The anti-proliferative effect of DFMO was specific since supplementation with polyamines reversed the effect of DFMO on proliferation and normalized cellular polyamine levels. Isometric force recordings in cultured rat tail artery rings showed that DFMO counteracts the decrease in contractility caused by culture with foetal bovine serum as growth stimulant. We conclude that inhibition of polyamine synthesis by DFMO may limit the first wave of cell proliferation and migration, which occurs in the acute phase after vascular injury. Besides its anti-proliferative effect, DFMO may prevent loss of the smooth muscle contractile phenotype in vascular injury. This article is protected by copyright. All rights reserved.
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43. |
- Grossi, Mario, et al.
(author)
-
Inhibition of polyamine uptake potentiates the anti-proliferative effect of polyamine synthesis inhibition and preserves the contractile phenotype of vascular smooth muscle cells.
- 2015
-
In: Journal of Cellular Physiology. - : Wiley. - 1097-4652 .- 0021-9541.
-
Journal article (peer-reviewed)abstract
- Increased vascular smooth muscle cell (VSMC) proliferation is a factor in atherosclerosis and injury-induced arterial (re)stenosis. Inhibition of polyamine synthesis by α-difluoro-methylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, attenuates VSMC proliferation with high sensitivity and specificity. However, cells can escape polyamine synthesis blockade by importing polyamines from the environment. To address this issue, polyamine transport inhibitors (PTIs) have been developed. We investigated the effects of the novel trimer44NMe (PTI-1) alone and in combination with DFMO on VSMC polyamine uptake, proliferation and phenotype regulation. PTI-1 efficiently inhibited polyamine uptake in primary mouse aortic and human coronary VSMCs in the absence as well as in the presence of DFMO. Interestingly, culture with DFMO for 2 days substantially (>95%) reduced putrescine (Put) and spermidine (Spd) contents without any effect on proliferation. Culture with PTI-1 alone had no effect on either polyamine levels or proliferation rate, but the combination of both treatments reduced Put and Spd levels below the detection limit and inhibited proliferation. Treatment with DFMO for a longer time period (4 days) reduced Put and Spd below their detection limits and reduced proliferation, showing that only a small pool of polyamines is needed to sustain VSMC proliferation. Inhibited proliferation by polyamine depletion was associated with maintained expression of contractile smooth marker genes. In cultured intact mouse aorta, PTI-1 potentiated the DFMO-induced inhibition of cell proliferation. The combination of endogenous polyamine synthesis inhibition with uptake blockade is thus a viable approach for targeting unwanted vascular cell proliferation in vivo, including vascular restenosis. This article is protected by copyright. All rights reserved.
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44. |
- Grossi, Mario, et al.
(author)
-
Vascular smooth muscle cell proliferation depends on caveolin-1-regulated polyamine uptake
- 2014
-
In: Bioscience Reports. - 0144-8463. ; 34, s. 729-741
-
Journal article (peer-reviewed)abstract
- Much evidence highlights the importance of polyamines for VSMC (vascular smooth muscle cell) proliferation and migration. Cav-1 (caveolin-1) was recently reported to regulate polyamine uptake in intestinal epithelial cells. The aim of the present study was to assess the importance of Cav-1 for VSMC polyamine uptake and its impact on cell proliferation and migration. Cav-1 KO (knockout) mouse aortic cells showed increased polyamine uptake and elevated proliferation and migration compared with WT (wild-type) cells. Both Cav-1 KO and WT cells expressed the smooth muscle differentiation markers SM22 and calponin. Cell-cycle phase distribution analysis revealed a higher proportion of Cav-1 KO than WT cells in the S phase. Cav-1 KO cells were hyper-proliferative in the presence but not in the absence of extracellular polyamines, and, moreover, supplementation with exogenous polyamines promoted proliferation in Cav-1 KO but not in WT cells. Expression of the solute carrier transporters Slc7a1 and Slc43a1 was higher in Cav-1 KO than in WT cells. ODC (ornithine decarboxylase) protein and mRNA expression as well as ODC activity were similar in Cav-1 KO and WT cells showing unaltered synthesis of polyamines in Cav-1 KO cells. Cav-1 was reduced in migrating cells in vitro and in carotid lesions in vivo. Our data show that Cav-1 negatively regulates VSMC polyamine uptake and that the proliferative advantage of Cav-1 KO cells is critically dependent on polyamine uptake. We provide proof-of-principle for targeting Cav-1-regulated polyamine uptake as a strategy to fight unwanted VSMC proliferation as observed in restenosis.
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45. |
- Grände, Gustaf, et al.
(author)
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Unaltered Size-selectivity of the Glomerular Filtration Barrier in Caveolin-1 Knock-out (KO) mice.
- 2009
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In: American Journal of Physiology: Renal, Fluid and Electrolyte Physiology. - : American Physiological Society. - 0363-6127. ; 297:2, s. 257-262
-
Journal article (peer-reviewed)abstract
- The transfer of albumin from blood to tissue has been found to be increased in caveolin-1 knock-out (KO) mice. This has been considered to reflect an increased microvascular permeability, conceivably caused by an increased endothelial production of nitric oxide (NO) in mice lacking caveolin-1. To investigate whether such an increase in endothelial NO-production would also affect the glomerular barrier characteristics, the glomerular sieving coefficients () to neutral, polydisperse fluorescein isothiocyanate (FITC)-Ficoll 70/400 (mol. radius 15-90 A) were determined in caveolin-1 KO mice vs. their wild-type counterparts. for Ficoll were assessed using high performance size exclusion chromatography (HPSEC) on blood and urine samples. Furthermore, the transcapillary escape rate (TER) of (125)I-labeled albumin and plasma volume (PV) were determined in both types of mice. Despite an increase in the glomerular filtration rate (GFR) in caveolin-1 KO mice (0.23+/-0.04 mL/min; n=7 vs. 0.10+/-0.02 mL/min; n=7; p<0.05) the glomerular Ficoll sieving curves were nearly identical. Furthermore, caveolin-1 KO mice showed an increased PV (6.59+/-0.42 mL/100g vs. 5.18+/-0.13 mL/100g; p<0.01) but only a tendency of an increased TER (14.69+/-1.59 %/h vs. 11.62+/-1.62 %/h; N.S.). It is concluded that in caveolin-1 KO mice the glomerular permeability was not increased, despite the presence of glomerular hyperfiltration. The present data are in line with the concept that the increased transvascular albumin leakage previously found in mice lacking caveolin-1 may be due to an elevation in systemic microvascular pressure following NO-induced precapillary vasodilatation, rather than being a consequence of an increased microvascular permeability per se. Key words: capillary permeability, nitric oxide, sieving coefficient, Ficoll, glomerular filtration rate.
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46. |
- Hansson, Björn, et al.
(author)
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A hypothesis for insulin resistance in primary human adipocytes involving MRTF-A and suppression of PPARγ
- 2020
-
In: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 533:1, s. 64-69
-
Journal article (peer-reviewed)abstract
- Obesity is the main risk factor behind insulin resistance and type 2 diabetes. Still, the mechanism behind adipocyte dysfunction is not yet resolved. Recently, we reported that rapid actin remodeling correlates with adipose cell size changes after short-term overfeeding. Therefore, we hypothesized that the actin-driven myocardin-related transcription factor (MRTF-A) contributes to impaired mature adipocyte function. Primary human adipocytes were subjected to adenoviral overexpression of MRTF-A or MRTF-B, followed by Western blot analysis and tracer glucose uptake assay. Further, we assessed cell size distribution, insulin response, MRTF-A localization, actin organization and degree of polymerization in adipocytes isolated from Ob/Ob mice. Overexpression of MRTF-A, but not MRTF-B, markedly suppressed PPARγ expression. Further, MRTF-A expression resulted in decreased IRS-1 level, shifted phosphorylation of Akt (pS473/pT308), IRS-1 (pS302) and AS160 (pT642), and lowered insulin-stimulated glucose uptake. Hypertrophic adipocytes from Ob/Ob mice displayed an increased proportion of polymerized actin, and increased nuclear translocation of MRTF-A compared with control (Ob/+). Similar with human adipocytes overexpressing MRTF-A, adipocytes isolated from Ob/Ob mice had reduced expression of IRS-1 and PPARγ, as well as impaired insulin response. Together, these data demonstrate that MRTF-A negatively influences insulin sensitivity and the expression of key targets in fully mature human adipocytes. This suggests that MRTF-A is poised to exert a transcriptional response in hypertrophic adipocytes, contributing to adipocyte dysfunction and insulin resistance.
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47. |
- Hansson, Björn, et al.
(author)
-
Rosiglitazone drives cavin-2/SDPR expression in adipocytes in a CEBPα-dependent manner
- 2017
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:3
-
Journal article (peer-reviewed)abstract
- Caveolae are abundant adipocyte surface domains involved in insulin signaling, membrane trafficking and lipid homeostasis. Transcriptional control mechanisms for caveolins and cavins, the building blocks of caveolae, are thus arguably important for adipocyte biology and studies in this area may give insight into insulin resistance and diabetes. Here we addressed the hypothesis that one of the less characterized caveolar components, cavin-2 (SDPR), is controlled by CCAAT/Enhancer Binding Protein (CEBPα) and Peroxisome Proliferator- Activated Receptor Gamma (PPARG). Using human mRNA expression data we found that SDPR correlated with PPARG in several tissues. This was also observed during differentiation of 3T3-L1 fibroblasts into adipocytes. Treatment of 3T3-L1-derived adipocytes with the PPARγ-activator Rosiglitazone increased SDPR and CEBPα expression at both the mRNA and protein levels. Silencing of CEBPα antagonized these effects. Further, adenoviral expression of PPARγ/CEBPα or Rosiglitazone-treatment increased SDPR expression in primary rat adipocytes. The myocardin family coactivator MKL1 was recently shown to regulate SDPR expression in human coronary artery smooth muscle cells. However, we found that actin depolymerization, known to inhibit MKL1 and MKL2, was without effect on SDPR mRNA levels in adipocytes, even though overexpression of MKL1 and MKL2 had the capacity to increase caveolins and cavins and to repress PPARγ/CEBPα. Altogether, this work demonstrates that CEBPα expression and PPARγ-activity promote SDPR transcription and further supports the emerging notion that PPARγ/CEBPα and MKL1/MKL2 are antagonistic in adipocytes.
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48. |
- He, Zhifei, et al.
(author)
-
CD14 is a co-receptor for TLR4 in the S100A9-induced pro-inflammatory response in monocytes
- 2016
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:5
-
Journal article (peer-reviewed)abstract
- The cytosolic Ca2+-binding S100A9 and S100A8 proteins form heterodimers that are primarily expressed in human neutrophils and monocytes. We have recently shown that S100A9 binds to TLR4 in vitro and induces TLR4-dependent NF-κB activation and a pro-inflammatory cytokine response in monocytes. In the present report we have further investigated the S100A9-mediated stimulation of TLR4 in monocytes. Using transmission immunoelectron microscopy, we detected focal binding of S100A9 to monocyte membrane subdomains containing the caveolin-1 protein and TLR4. Furthermore, the S100A9 protein was detected in early endosomes of the stimulated cells, indicating that the protein could be internalized by endocytosis. Although stimulation of monocytes with S100A9 was strictly TLR4-dependent, binding of S100A9 to the plasma membrane and endocytosis of S100A9 was still detectable and coincided with CD14 expression in TLR4-deficient cells. We therefore investigated whether CD14 would be involved in the TLR4-dependent stimulation and could show that the S100A9-induced cytokine response was inhibited both in CD14-deficient cells and in cells exposed to CD14 blocking antibodies. Further, S100A9 was not internalized into CD14-deficient cells suggesting a direct role of CD14 in endocytosis of S100A9. Finally, we could detect satiable binding of S100A9 to CD14 in surface plasmon resonance experiments. Taken together, these results indicate that CD14 is a co-receptor of TLR4 in the S100A9-induced cytokine response.
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49. |
- Hien Tran, Thi, et al.
(author)
-
Elevated glucose levels promote contractile and cytoskeletal gene expression in vascular smooth muscle via Rho/protein kinase C and actin polymerization.
- 2016
-
In: Journal of Biological Chemistry. - 1083-351X. ; 291:7, s. 68-3552
-
Journal article (peer-reviewed)abstract
- Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hyper-contractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, qPCR and western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers was increased in isolated smooth muscle cells cultured under high compared to low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization and myocardin related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility.
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50. |
- Karbalaei, Mardjaneh, et al.
(author)
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Detrusor Induction of miR-132/212 following Bladder Outlet Obstruction: Association with MeCP2 Repression and Cell Viability.
- 2015
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:1
-
Journal article (peer-reviewed)abstract
- The microRNAs (miRNAs) miR-132 and miR-212 have been found to regulate synaptic plasticity and cholinergic signaling and recent work has demonstrated roles outside of the CNS, including in smooth muscle. Here, we examined if miR-132 and miR-212 are induced in the urinary bladder following outlet obstruction and whether this correlates with effects on gene expression and cell growth. Three to seven-fold induction of miR-132/212 was found at 10 days of obstruction and this was selective for the detrusor layer. We cross-referenced putative binding sites in the miR-132/212 promoter with transcription factors that were predicted to be active in the obstruction model. This suggested involvement of Creb and Ahr in miR-132/212 induction. Creb phosphorylation (S-133) was not increased, but the number of Ahr positive nuclei increased. Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells. To identify miR-132/212 targets, we correlated the mRNA levels of validated targets with the miRNA levels. Significant correlations between miR-132/212 and MeCP2, Ep300, Pnkd and Jarid1a were observed, and the protein levels of MeCP2, Pnkd and Ache were reduced after obstruction. Reduction of Ache however closely matched a 90% reduction of synapse density arguing that its repression was unrelated to miR-132/212 induction. Importantly, transfection of antimirs and mimics in cultured detrusor cells increased and decreased, respectively, the number of cells and led to changes in MeCP2 expression. In all, these findings show that obstruction of the urethra increases miR-132 and miR-212 in the detrusor and suggests that this influences gene expression and limits cell growth.
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