| 1. |
- Alajbegovic, Azra, et al.
(författare)
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Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization
- 2017
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Ingår i: Biochimica et Biophysica Acta - Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1864:6, s. 1088-1098
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Bhattachariya, Anirban, et al.
(författare)
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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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Ingår i: Cardiovascular Research. - : Oxford University Press (OUP). - 1755-3245 .- 0008-6363. ; 101:2, s. 288-296
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Bhattachariya, Anirban, et al.
(författare)
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PYK2 selectively mediates signals for growth versus differentiation in response to stretch of spontaneously active vascular smooth muscle.
- 2014
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Ingår i: Physiological Reports. - : Wiley. - 2051-817X. ; 2:7
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Tidskriftsartikel (refereegranskat)abstract
- Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium-dependent signals. We studied the role of the calcium- and integrin-activated proline-rich tyrosine kinase 2 (PYK2) in stretch-induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr-402 was increased both by a 10-min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF-4594755 (0.5-1 μmol/L), while still sensitive to stretch. In 3-day organ culture, PF-4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high-K(+) (60 mmol/L) or to α1-adrenergic stimulation by cirazoline. Basal and stretch-induced PYK2 phosphorylation in culture were inhibited by PF-4594755, closely mimicking inhibition of non-voltage-dependent calcium influx by 2-APB (30 μmol/L). In contrast, the L-type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch-induced but not basal PYK2 phosphorylation. Stretch-induced Akt and ERK1/2 phosphorylation was eliminated by PF-4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch-sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 μmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage- and non-voltage-dependent calcium influx. A small-molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle.
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| 4. |
- Dahan, Diana, et al.
(författare)
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Induction of angiotensin converting enzyme after miR-143/145 deletion is critical for impaired smooth muscle contractility.
- 2014
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Ingår i: American Journal of Physiology: Cell Physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 307:12, s. 1093-1101
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Forte, Amalia, et al.
(författare)
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Local inhibition of ornithine decarboxylase reduces vascular stenosis in a murine model of carotid injury
- 2013
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Ingår i: International Journal of Cardiology. - : Elsevier BV. - 0167-5273. ; 168:4, s. 3370-3380
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re) stenosis. Methods: The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd. 3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression. Results: DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd. 3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury. Conclusions: Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process. (C) 2013 Elsevier Ireland Ltd. All rights reserved.
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| 6. |
- Grossi, Mario, et al.
(författare)
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Inhibition of Polyamine Formation Antagonizes Vascular Smooth Muscle Cell Proliferation and Preserves the Contractile Phenotype.
- 2014
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Ingår i: Basic & Clinical Pharmacology & Toxicology. - : Wiley. - 1742-7843 .- 1742-7835. ; 115:5, s. 379-388
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Grossi, Mario, et al.
(författare)
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Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle
- 2017
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Ingår i: Journal of Cellular Physiology. - : Wiley. - 0021-9541. ; 232:11, s. 3088-3102
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Tidskriftsartikel (refereegranskat)abstract
- Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24–96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.
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| 8. |
- Hien Tran, Thi, et al.
(författare)
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Elevated glucose levels promote contractile and cytoskeletal gene expression in vascular smooth muscle via Rho/protein kinase C and actin polymerization.
- 2016
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 291:7, s. 68-3552
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Tidskriftsartikel (refereegranskat)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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| 9. |
- Madsen, Marie, et al.
(författare)
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Uremia modulates the phenotype of aortic smooth muscle cells
- 2017
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Ingår i: Atherosclerosis. - : Elsevier BV. - 0021-9150. ; 257, s. 64-70
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Tidskriftsartikel (refereegranskat)abstract
- Background and aims Chronic kidney disease leads to uremia and markedly accelerates atherosclerosis. Phenotypic modulation of smooth muscle cells (SMCs) in the arterial media plays a key role in accelerating atherogenesis. The aim of this study was to investigate whether uremia per se modulates the phenotype of aortic SMCs in vivo. Methods Moderate uremia was induced by 5/6 nephrectomy in apolipoprotein E knockout (ApoE-/-) and wildtype C57Bl/6 mice. Plasma analysis, gene expression, histology, and myography were used to determine uremia-mediated changes in the arterial wall. Results Induction of moderate uremia in ApoE-/- mice increased atherosclerosis in the aortic arch en face 1.6 fold (p = 0.04) and induced systemic inflammation. Based on histological analyses of aortic root sections, uremia increased the medial area, while there was no difference in the content of elastic fibers or collagen in the aortic media. In the aortic arch, mRNA and miRNA expression patterns were consistent with a uremia-mediated phenotypic modulation of SMCs; e.g. downregulation of myocardin, α-smooth muscle actin, and transgelin; and upregulation of miR146a. Notably, these expression patterns were observed after acute (2 weeks) and chronic (19 and 30 weeks) uremia, both under normo- and hypercholesterolemic settings. Functionally, aortic constriction was decreased in uremic as compared to non-uremic aorta segments, as measured by myography. Conclusions Uremia modulates the phenotype of aortic SMCs as determined by mRNA/miRNA expression, an increased medial area, and decreased aortic contractility. We propose that this phenotypic modulation of SMCs precedes the acceleration of atherosclerosis observed in uremic mice.
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| 10. |
- Svensson, Daniel, et al.
(författare)
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Inhibition of MicroRNA-125a Promotes Human Endothelial Cell Proliferation and Viability through an Antiapoptotic Mechanism.
- 2014
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Ingår i: Journal of Vascular Research. - : S. Karger AG. - 1423-0135 .- 1018-1172. ; 51:3, s. 239-245
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Tidskriftsartikel (refereegranskat)abstract
- The microRNA-125a (miR-125a) is highly expressed in endothelial cells, but its role in vascular biology is not known. Endothelial cell proliferation and viability play an important role in endothelial healing, and we hypothesize that miR-125a regulates this process. The aim of the present study was to investigate if miR-125a controls human endothelial cell proliferation, viability and endothelial healing, and to assess the mechanisms involved. We showed that overexpression of miR-125a by transfection with miR-125a mimic reduced human umbilical vein endothelial cell (HUVEC) proliferation and viability, and stimulated apoptosis as demonstrated by a miR-125a-induced increase of the proportion of annexin V-positive cells monitored by flow cytometry. Moreover, we showed that the miR-125a mimic downregulated the antiapoptotic Bcl2 protein and upregulated caspase 3, suggesting that these two proteins represent molecular targets for miR-125a. Accordingly, transfection with miR-125a inhibitor, downregulating miR-125a expression, promoted HUVEC proliferation and viability, and reduced apoptosis. Importantly, transfection with miR-125a inhibitor promoted HUVEC tube formation in Matrigel, suggesting that reduction of miR-125a has a proangiogenic effect. In conclusion, downregulation of miR-125a through local transfection with miR-125a inhibitor might be a new way to enhance endothelial cell proliferation and viability, thereby promoting the reendothelialization observed in response to intimal injury. © 2014 S. Karger AG, Basel.
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