| 1. |
- 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
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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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| 2. |
- Albinsson, Sebastian, et al.
(author)
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Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization
- 2004
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In: Journal of Biological Chemistry. - 1083-351X. ; 279:33, s. 34849-34855
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Journal article (peer-reviewed)abstract
- Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system (alpha-actin, calponin, SM22alpha, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins (beta-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins beta-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22alpha synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, > 15 min; cofilin-P, > 1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.
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| 3. |
- Berglund, Lisa, et al.
(author)
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Novel blocker of NFAT activation inhibits IL-6 production in human myometrial arteries and reduces vascular smooth muscle cell proliferation
- 2007
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In: American Journal of Physiology: Cell Physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 292:3, s. 1167-1178
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Journal article (peer-reviewed)abstract
- The calcineurin/nuclear factor of activated T cells ( NFAT) signaling pathway has been found to play a role in regulating growth and differentiation in several cell types. However, the functional significance of NFAT in the vasculature is largely unclear. Here we show that NFATc1, NFATc3, and NFATc4 are expressed in human myometrial arteries. Confocal immunofluorescence and Western blot analysis revealed that endothelin-1 efficiently increases NFATc3 nuclear accumulation in native arteries. Endothelin-1 also stimulates NFAT-dependent transcriptional activity, as shown by a luciferase reporter assay. Both the agonist-induced NFAT nuclear accumulation and transcriptional activity were prevented by the calcineurin inhibitor CsA and by the novel NFAT blocker A-285222. Chronic inhibition of NFAT significantly reduced IL-6 production in intact myometrial arteries and inhibited cell proliferation in vascular smooth muscle cells cultured from explants from the same arteries. Furthermore, by using small interfering RNA-mediated reduction of NFATc3, we show that this isoform is involved in the regulation of cell proliferation. Protein synthesis in intact arteries was investigated using autoradiography of [S-35] methionine incorporation in serum-free culture. Inhibition of NFAT signaling did not affect overall protein synthesis or specifically the synthesis rates of major proteins associated with the contractile/cytoskeletal system. An intact contractile phenotype under these conditions was also shown by unchanged force response to depolarization or agonist stimulation. Our results demonstrate NFAT expression and activation in native human vessels and point out A-285222 as a powerful pharmacological blocker of NFAT signaling in the vasculature.
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| 4. |
- Bhattachariya, Anirban, et al.
(author)
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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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In: Physiological Reports. - : Wiley. - 2051-817X. ; 2:7
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Journal article (peer-reviewed)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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| 5. |
- Dreja, Karl, et al.
(author)
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Rat arterial smooth muscle devoid of ryanodine receptor function: effects on cellular Ca2+ handling
- 2001
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In: British Journal of Pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 132:8, s. 1957-1966
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Journal article (peer-reviewed)abstract
- The roles of intracellular Ca2+ stores and ryanodine (Ry) receptors for vascular Ca2+ homeostasis and viability were investigated in rat tail arterial segments kept in organ culture with Ry (10 100 M) for up to 4 days. Acute exposure to Ry or the non-deactivating ryanodine analogue C10-Oeq glycyl ryanodine (10 M) eliminated Ca2+ release responses to caffeine (20 mM) and noradrenaline (NA, 10 M), whereas responses to NA, but not caffeine, gradually returned to normal within 4 days of exposure to Ry. Ry receptor protein was detected on Western blots in arteries cultured either with or without Ry. Brief Ca2+ release events (sparks) were absent after culture with Ry, whereas Ca2+ waves still occurred. The propagation velocity of waves was equal (19 m s-1) in tissue cultured either with or without Ry. Inhibition of Ca2+ accumulation into the sarcoplasmic reticulum (SR) by culture with caffeine (5 mM), cyclopiazonic acid or thapsigargin (both 10 M) decreased contractility due to Ca2+-induced cell damage. In contrast, culture with Ry did not affect contractility. Removal of Ca2+ from the cytosol following a Ca2+ load was retarded after Ry culture. Thapsigargin reduced the rate of Ca2+ removal in control cultured rings, but had no effect after Ry culture. It is concluded that intracellular Ca2+ stores recover during chronic Ry treatment, while Ry receptors remain non-functional. Ry receptor activity is required for Ca2+ sparks and for SR-dependent recovery from a Ca2+ load, but not for Ca2+ waves or basal Ca2+ homeostasis.
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| 6. |
- Forte, Amalia, et al.
(author)
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Injury to rat carotid arteries causes time-dependent changes in gene expression in contralateral uninjured arteries
- 2009
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In: Clinical Science. - 1470-8736. ; 116:1-2, s. 125-136
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Journal article (peer-reviewed)abstract
- Vascular surgery aimed at stenosis removal induces local reactions often leading to restenosis. Although extensive analysis has been focused on pathways activated in injured arteries, little attention has been devoted to associated systemic vascular reactions. The aim of the present study was to analyse changes occurring in contralateral uninjured rat carotid arteries in the acute phase following unilateral injury. WKY (Wistar-Kyoto) rats were subjected to unilateral carotid arteriotomy. Contralateral uninjured carotid arteries were harvested from 4 h to 7 days after injury. Carotid arteries were also harvested from sham-operated rats and uninjured rats. Carotid morphology and morphometry were examined. Affymetrix microarrays were used for differential analysis of gene expression. A subset of data was validated by real-time RT-PCR (reverse transcription-PCR) and verified at the protein level by Western blotting. A total of 1011 genes were differentially regulated in contralateral uninjured carotid arteries from 4 h to 7 days after arteriotomy (P < 0.0001; fold change, >= 2) and were classified into 19 gene ontology functional categories. To a lesser extent, mRNA variations also occurred in carotid arteries of sham-operated rats. Among the changes, up-regulation of members of the RAS (renin-angiotensin system) was detected, with possible implications for vasocompensative mechanisms induced by arteriotomy. In particular, a selective increase in the 69 kDa isoform of the N-domain of ACE (angiotensin-converting enzyme), and not the classical somatic 195 kDa isoform, was observed in contralateral uninjured carotid arteries, suggesting that this 69 kDa isoenzyme could influence local Angll (angiotensin II) production. In conclusion, systemic reactions to injury occur in the vasculature, with potential clinical relevance, and suggest that caution is needed in the choice of controls during experimental design in vivo.
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| 7. |
- Grossi, Mario, et al.
(author)
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Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle
- 2017
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In: Journal of Cellular Physiology. - : Wiley. - 0021-9541. ; 232:11, s. 3088-3102
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Journal article (peer-reviewed)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. |
- Hellstrand, Per, et al.
(author)
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Cross-bridge kinetics during shortening in early and sustained contraction of intestinal smooth muscle
- 1993
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In: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 265:3 Pt 1, s. 695-703
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Journal article (peer-reviewed)abstract
- Mechanisms responsible for the decrease in shortening velocity after prolonged contraction ("latch" state) were investigated at identical force during early (20 s, "phasic") and sustained (5 min, "tonic") phases of high-K+ (25-30 mM) contractions in smooth muscle of guinea pig taenia coli. Cytoplasmic Ca2+ concentration, myosin light-chain phosphorylation, and maximum shortening velocity all declined from 20 s to 5 min of contraction. The time course of shortening following isotonic quick release was biexponential, with a fastest rate constant of approximately 80 s-1 in both phasic and tonic contractions. Stiffness was identical in phasic and tonic contraction; however, after a release to slack length and unloaded shortening, stiffness during restretch was greater in tonic contraction (51 vs. 43% of isometric stiffness after 16 ms of unloaded shortening). Stiffness decreased after release with a rate constant of approximately 200 s-1, slightly greater in phasic than in tonic contraction. The results indicate that the number of attached cross bridges during unloaded shortening, while substantially reduced relative to the isometric value, is higher in latch than in nonlatch, consistent with a lower detachment relative to attachment rate.
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| 9. |
- Holm, Anders, et al.
(author)
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Down-regulation of endothelial cell estrogen receptor expression by the inflammation promoter LPS.
- 2010
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In: Molecular and Cellular Endocrinology. - : Elsevier BV. - 1872-8057 .- 0303-7207. ; 319, s. 8-13
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Journal article (peer-reviewed)abstract
- Endothelial cells express both estrogen receptor (ER) alpha and beta. The objective of this study was to investigate if and how mediators of inflammation regulate endothelial cell ERalpha and ERbeta expression. ERalpha and ERbeta transcript and protein expression were determined by real-time quantitative PCR and Western blotting, respectively, in endothelial cell line bEnd.3 cells stimulated with the inflammation promoter lipopolysaccharide (E. coli LPS). Stimulation with LPS (500ng/ml and 10mug/ml) for 4 days reduced both ERalpha and ERbeta mRNA levels. The glucocorticoid dexamethasone (1muM) had no effect on LPS-induced attenuation of ERalpha and beta transcript expression. Full-length 66-67kDa ERalpha protein was unaffected by 4 days stimulation with LPS, while the 46-kDa ERalpha isoform was reduced by about 20%. ERbeta protein was reduced by about 40% by LPS at 4 days. Treatment with 17beta-estradiol (E(2), 100nM) for 4 days increased ERbeta mRNA by about 8 times but had no effect on ERalpha mRNA level. The E(2)-induced increase in ERbeta transcript was not associated with increased ERbeta protein. E(2) increased ERbeta mRNA expression also in the presence of LPS, suggesting that inflammation-induced impairment of ERbeta signalling is rescued by estrogen.
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| 10. |
- Lindqvist, Anders, et al.
(author)
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Long-term effects of Ca(2+) on structure and contractility of vascular smooth muscle
- 1999
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In: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 277:1, s. 64-73
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Journal article (peer-reviewed)abstract
- Culture of dispersed smooth muscle cells is known to cause rapid modulation from the contractile to the synthetic cellular phenotype. However, organ culture of smooth muscle tissue, with maintained extracellular matrix and cell-cell contacts, may facilitate maintenance of the contractile phenotype. To test the influence of culture conditions, structural, functional, and biochemical properties of rat tail arterial rings were investigated after culture. Rings were cultured for 4 days in the absence and presence of 10% FCS and then mounted for physiological experiments. Intracellular Ca(2+) concentration ([Ca(2+)](i)) after stimulation with norepinephrine was similar in rings cultured with and without FCS, whereas force development after FCS was decreased by >50%. The difference persisted after permeabilization with beta-escin. These effects were associated with the presence of vasoconstrictors in FCS and were dissociated from its growth-stimulatory action. FCS treatment increased lactate production but did not affect ATP, ADP, or AMP contents. The contents of actin and myosin were decreased by culture but similar for all culture conditions. There was no effect of FCS on calponin contents or myosin SM1/SM2 isoform composition, nor was there any appearance of nonmuscle myosin. FCS-stimulated rings showed evidence of cell degeneration not found after culture without FCS or with FCS + verapamil (1 microM) to lower [Ca(2+)](i). The decreased force-generating ability after culture with FCS is thus associated with increased [Ca(2+)](i) during culture and not primarily caused by growth-associated modulation of cells from the contractile to the synthetic phenotype.
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