Spontaneously Hypertensive Rats Vascular Smooth Muscle Cells Research Articles

Exosome-Mediated Transfer of ACE (Angiotensin-Converting Enzyme) From Adventitial Fibroblasts of Spontaneously Hypertensive Rats Promotes Vascular Smooth Muscle Cell Migration.

Migration of vascular smooth muscle cells (VSMCs) is pivotal for vascular remodeling in hypertension. Vascular adventitial fibroblasts (AFs) are important in the homeostasis of vascular structure. This study is designed to investigate the roles of AF exosomes (AFE) in VSMC migration and underling mechanism. Primary VSMCs and AFs were obtained from the aorta of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. VSMC migration was evaluated with Boyden chamber assay and wound healing assay. AFE from WKY rats and SHR were isolated and identified. AFE from SHR promoted but AFE from WKY rats had no significant effect on VSMC migration. The effects of AFE on VSMC migration were prevented by an exosome inhibitor GW4869, an AT1R (Ang II [angiotensin II] type 1 receptor) antagonist losartan, or an inhibitor of ACE (angiotensin-converting enzyme) captopril. ACE contents and activity were much higher in AFE from SHR than those from WKY rats. There were no significant difference in Ang II and AT1R mRNA and protein levels between AFE from SHR and AFE from WKY rats. AFE from SHR increased Ang II and ACE contents and ACE activity in VSMCs of WKY rats and SHR. The changes of Ang II contents and ACE activity were prevented by captopril. ACE knockdown in AFs reduced ACE contents and activity in AFE from SHR and inhibited AFE-induced migration of VSMCs of WKY rats and those of SHR. These results indicate that exosomes from AFs of SHR transfer ACE to VSMCs, which increases Ang II levels and activates AT1R in VSMCs and thereby promotes VSMC migration.

Glucose-regulated protein 78 in lipid rafts elevates vascular smooth muscle cell proliferation of spontaneously hypertensive rats by controlling platelet-derived growth factor receptor signaling.

The multifunctional glucose-regulated protein 78 (GRP78) is known to be differentially expressed in the lipid rafts of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) and normal Wistar-Kyoto (WKY) rats. However, its role in VSMCs from SHRs remains to be elucidated. This work was conducted to investigate the contribution made by GRP78 in VSMCs. GRP78 expression in VSMC lipid rafts decreased in WKY rats with age, but not in SHRs. Transfection with GRP78-siRNA attenuated not only platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and aortic sprout outgrowth but also the phosphorylation of PDGF receptor (PDGFR)-β, Akt, and extracellular signal-regulated kinase (Erk) 1/2 in VSMCs in response to PDGF-BB. Moreover, GRP78 knockdown also reduced the PDGF-BB-induced dimerization of PDGFR-β and GRP78 in SHR VSMCs. The phosphorylation of GRP78 and PDGFR-β was elevated in VSMCs treated with PDGF-BB and was completely abolished by AG1296 (a PDGFR inhibitor). Moreover, the binding of PDGFR-β to GRP78 and the co-localization of GRP78 to PDGFR-β in VSMCs were stronger in SHRs than in WKY rat controls. This study demonstrates that the PDGF-BB-induced proliferation of SHR VSMCs is mediated by the expressional upregulation of GRP78 on VSMC lipid rafts in SHRs, probably via the regulation of PDGFR-β-GRP78 binding and their cross-activation. These observations indicate that GRP78 may play important roles in the pathological progression of SHR VSMCs.

Sulfatase 1 mediates the attenuation of Ang II-induced hypertensive effects by CCL5 in vascular smooth muscle cells from spontaneously hypertensive rats

Extracellular sulfatases, sulfatase 1 (Sulf1) and sulfatase 2 (Sulf2), play a pivotal role in cell signaling and carcinogenesis. Chemokine CCL5 inhibits Ang II-induced hypertensive mediators via angiotensin II (Ang II) type 2 receptor (AT2 R) pathway in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). In this study, we investigated the effect of Sulfs on anti-hypertensive effects of CCL5 in SHR VSMCs. CCL5 attenuated Ang II-induced inhibition of sulfatase activity in SHR VSMCs. Inhibition of Ang II-induced 12-lipoxygenase (12-LO) and endothelin-1 (ET-1) expression by CCL5 was reduced in Sulf1 small interfering RNA (siRNA)-transfected SHR VSMCs. In addition, attenuation of Ang II-induced dimethylarginine dimethylaminohydrolase-1 (DDAH-1) inhibition by CCL5 was reduced in Sulf1 siRNA-transfected SHR VSMCs. Downregulation of Sulf2 did not affect inhibitory effects of CCL5 on Ang II-induced 12-LO and ET-1 expression and Ang II-induced inhibition of DDAH-1 expression in SHR VSMCs. Downregulation of Sulf1 abrogated the expression of CCL5-induced AT2 R messenger RNA (mRNA) and synergistic effect of CCL5 on Ang II-induced AT2 R expression in SHR VSMCs. These findings suggest that Sulf1 is a potential up-regulatory factor in anti-hypertensive actions of CCL5 via AT2 R pathway on Ang II-induced hypertensive effects in SHR VSMCs.

Nesfatin-1 functions as a switch for phenotype transformation and proliferation of VSMCs in hypertensive vascular remodeling

The phenotypic transformation from differentiated to dedifferentiated vascular smooth muscle cells (VSMCs) plays a crucial role in VSMC proliferation and vascular remodeling in many cardiovascular diseases including hypertension. Nesfatin-1, a multifunctional adipocytokine, is critically involved in the regulation of blood pressure. However, it is still largely unexplored whether nesfatin-1 is a potential candidate in VSMC phenotypic switch and proliferation in hypertension. Experiments were carried out in Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), human VSMCs and primary rat aortic VSMCs. We showed that the expression of nesfatin-1 was upregulated in media layer of the aorta in SHR and SHR-derived VSMCs. Nesfatin-1 promoted VSMC phenotypic transformation, accelerated cell cycle progression and proliferation. Knockdown of nesfatin-1 inhibited the VSMC phenotype switch from a contractile to a synthetic state, attenuated cell cycle progression and retarded VSMC proliferation in SHR-derived VSMCs. Moreover, nesfatin-1-activated PI3K/Akt/mTOR signaling was abolished by JAK/STAT inhibitor WP1066, and the increased phosphorylation levels of JAK2/STAT3 in response to nesfatin-1 were suppressed by inhibition of PI3K/Akt/mTOR in VSMCs. Pharmacological blockade of the forming feedback loop between PI3K/Akt/mTOR and JAK2/STAT3 prevented the proliferation of nesfatin-1-incubated VSMCs and primary VSMCs from SHR. Chronic intraperitoneal injection of nesfatin-1 caused severe hypertension and cardiovascular remodeling in normal rats. In contrast, silencing of nesfatin-1 gene ameliorated hypertension, phenotype switching, and vascular remodeling in the aorta of SHR. Therefore, our data identified nesfatin-1 as a key modulator in hypertension and vascular remodeling by facilitating VSMC phenotypic switching and proliferation.

Vascular smooth muscle cells direct extracellular dysregulation in aortic stiffening of hypertensive rats.

SummaryAortic stiffening is an independent risk factor that underlies cardiovascular morbidity in the elderly. We have previously shown that intrinsic mechanical properties of vascular smooth muscle cells (VSMCs) play a key role in aortic stiffening in both aging and hypertension. Here, we test the hypothesis that VSMCs also contribute to aortic stiffening through their extracellular effects. Aortic stiffening was confirmed in spontaneously hypertensive rats (SHRs) vs. Wistar‐Kyoto (WKY) rats in vivo by echocardiography and ex vivo by isometric force measurements in isolated de‐endothelized aortic vessel segments. Vascular smooth muscle cells were isolated from thoracic aorta and embedded in a collagen I matrix in an in vitro 3D model to form reconstituted vessels. Reconstituted vessel segments made with SHR VSMCs were significantly stiffer than vessels made with WKY VSMCs. SHR VSMCs in the reconstituted vessels exhibited different morphologies and diminished adaptability to stretch compared to WKY VSMCs, implying dual effects on both static and dynamic stiffness. SHR VSMCs increased the synthesis of collagen and induced collagen fibril disorganization in reconstituted vessels. Mechanistically, compared to WKY VSMCs, SHR VSMCs exhibited an increase in the levels of active integrin β1‐ and bone morphogenetic protein 1 (BMP1)‐mediated proteolytic cleavage of lysyl oxidase (LOX). These VSMC‐induced alterations in the SHR were attenuated by an inhibitor of serum response factor (SRF)/myocardin. Therefore, SHR VSMCs exhibit extracellular dysregulation through modulating integrin β1 and BMP1/LOX via SRF/myocardin signaling in aortic stiffening.

Abstract 73: Pre- and Post-Translational Regulation of Lysyl Oxidase/β1-Intergrin in Aortic Vascular Smooth Cell of Hypertension

Background: An increase in aortic stiffness is a fundamental component of hypertension . We previous showed that aortic vascular smooth muscle cells (VSMCs) play an causative role in aortic stiffening not only due to the increased intrinsic stiffness in VSMC itself but also due to its interaction with extracellular matrix (ECM). However, the molecular mechanism involved is unclear. Here, we test the hypothesis that the aortic VSMCs mediates pre- and post-translational regulation of Lysyl Oxidase (Lox)/β1intergrain pathway which may be one of the mechanisms of VSMCs-ECM remodeling in hypertension. Methods and Results: 4 months old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Aortic pressure and stiffness were measured with an invasive catheter and Doppler imaging echocardiography, respectively. Compared to WKY, SHR showed a significant higher aortic pressure (mean arterial pressure (MAP) 138.7±11.8 vs 102.7±7 mmHg, ( p <0.01) and a greater aorta stiffness reflected by 2.5-fold higher arterial stiffness index (ASI) in SHR vs WKY ( p <0.01). VSMCs were isolated from thoracic aorta, then cultured and measured at passages 2 to 4. Real time PCR and western blot were used to detect the gene and protein expression. Compared to WKY, VSMCs from SHR showed a significant increase in mRNA Lox, a copper-dependent amine oxidase mediating the crosslinking of collagens and elastin (2.7 folds vs WKY, p <0.01). The abundance of LOX protein was significant increased not only at pro-enzyme lox level, but also at the regulatory pro-peptide lox and matured active enzyme Lox in SHR VSMCs by 3.5 folds and 1.4 folds respectively ( p <0.01), indicating an increased post-translational regulation of LOX in SHR VSMCs. In addition, the glycosylated β1 integrin, a transmembrane receptor that facilitate cell-ECM adhesion, was found to be significant increase in SHR VSMCs vs WKY. Conclusion: The regulation of pre- and post-translational regulation of Lox/β1intergrain pathway in aortic VSMCs presents a novel mechanism of cell-ECM interaction, which may contribute to the increased aortic stiffness in SHR.

Sulfatase 1 mediates the inhibitory effect of angiotensin II type 2 receptor inhibitor on angiotensin II-induced hypertensive mediator expression and proliferation in vascular smooth muscle cells from spontaneously hypertensive rats

Background: Extracellular sulfatases (Sulfs), sulfatase 1 (Sulf1) and sulfatase 2 (Sulf2), play a pivotal role in cell signaling by remodeling the 6-O-sulfation of heparan sulfate proteoglycans on the cell surface. The present study examined the effects of Sulfs on angiotensin II (Ang II)-induced hypertensive mediator expre-ssion and vascular smooth muscle cells (VSMCs) proliferation in spontaneously hypertensive rats (SHR). Methods: Ang II receptors, 12-lipoxygenase (12-LO), and endothelin-1 (ET-1) messenger RNA (mRNA) ex-pressions in SHR VSMCs were analyzed by real-time polymerase chain reaction and Western blotting. VSMCs proliferation was determined by [ 3 H]-thymidine incorporation. Results: Basal Sulfs mRNAs expression and enzyme activity were elevated in SHR VSMCs. However, Sulfs had no effect on the basal or Ang II-induced 12-LO and ET-1 mRNA expression in SHR VSMCs. The inhibition of Ang II-induced 12-LO and ET-1 expression by blockade of the Ang II type 2 receptor (AT 2 R) pathway was not observed in Sulf1 siRNA-transfected SHR VSMCs. However, Sulf2 did not affect the action of AT 2 R inhibitor on Ang II-induced 12-LO and ET-1 expression in SHR VSMCs. The down-regulation of Sulf1 induced a reduction of AT 2 R mRNA expression in SHR VSMCs. In addition, the inhibition of Ang II-induced VSMCs proliferation by blockade of the AT 2 R pathway was mediated by Sulf1 in SHR VSMCs. Conclusion: These findings suggest that extracellular sulfatase Sulf1 plays a modulatory role in the AT 2 R pathway that leads to an Ang II-induced hypertensive effects in SHR VSMCs.

Abstract 188: I X2 X2

Background: An increase in aortic stiffness is a fundamental component of hypertension. However, the molecular mechanism involved is unclear. Our hypothesis is that the increased aortic stiffness in hypertension is partially due to the activation of serum response factor (SRF) /myocardin in vascular smooth muscle cells (VSMCs). Methods: 4 months old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Aortic pressure and stiffness were measured by a Millar catheter and by Doppler imaging echocardiography, respectively. VSMCs were isolated from thoracic aorta (TA), then cultured and tested at passages 2 to 4. Real time PCR and western blot were used to detect the gene and protein expression. Results: Aortic pressure was higher in SHR than WKY (mean arterial pressure (MAP) 138.7±11.8 vs 102.7±7 mmHg, p <0.01). Aorta stiffness reflected by arterial stiffness index (ASI) was 2.5-fold higher in SHR vs WKY ( p <0.01). Compared to WKY, VSMCs from SHR TA showed a 12-fold increase in SRF, a nuclear transcriptional factor, and 2.7-fold increase in myocardin, a smooth muscle specific coactivator of SRF, at both mRNA and protein levels (All, p <0.01 vs. WKY TA VSMCs). Myocardin nuclear translocation was also markedly increased in TA VSMCs from SHR by 12-fold versus WKY, ( p <0.01). SRF/myocardin downstream target genes, α-smooth muscle actin (α-SMA) and SM22 were increased by 3-fold in TA VSMCs in SHR vs WKY ( p <0.01). In vitro , CCG-100602, a specific inhibitor of SRF/myocardin interaction, not only strikingly repressed the increase of the expression in SRF, myocardin and the downstream targets α-SMA and SM22, but also blocked the nuclear translocation of myocardin in TA VSMCs from SHR ( p <0.01 vs untreated SHR VSMCs). However, such effects were not observed on WKY TA VSMCs. In vivo , 2-week treatment with CCG-100602 significantly reduced MAP and ASI by 19% and 63% in SHR, respectively ( p <0.01 vs untreated SHR), while aortic MAP and ASI in WKY rats remain unchanged upon the treatments. Conclusion: The activation of myocardin/SRF in aortic VSMCs presents a novel mechanism of increased aortic stiffness in SHR.

Farnesyl pyrophosphate synthase inhibitor, ibandronate, improves endothelial function in spontaneously hypertensive rats.

Reactive oxygen species (ROS), originating predominantly from vascular smooth muscle cells (VSMCs), lead to vascular damage and endothelial dysfunction in rats with hypertension. The downstream signaling pathways of farnesyl pyrophosphate (FPP) synthase, Ras-related C3 botulinum toxin substrate 1 (Rac1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mediate the generation of ROS. The present study investigated the effect of the FPP synthase inhibitor, ibandronate, on ROS production, the possible beneficial effect on endothelial dysfunction and the underlying mechanisms in spontaneously hypertensive rats (SHRs). The SHRs were treated with ibandronate for 30 days. Endothelium-dependent and independent vasorelaxation were measured in isolated aortic rings. Additionally, VSMCs from the SHRs and Wistar-Kyoto (WKY) rats were cultured. The production of ROS and activation of NADPH oxidase were determined using fluorescence and chemiluminescence, respectively, in vivo and in vitro. Angiotensin II (Ang II) increased ROS production in the cultured VSMCs from the WKY rats and SHRs, in a concentration-dependent manner. The Ang II-induced responses were more marked in the SHR VSMCs, compare with those in the WKY VSMCs, however, the response decreased significantly following ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, translocation of NADPH oxidase subunit p47phox, and activities of NADPH oxidase and Rac1 in the aorta and VSMCs, and improved the impaired endothelium-dependent vasodilation in the SHRs. Adding geranylgeraniol, but not farnesol or mevalonate, reversed the inhibitory effects of ibandronate. In addition, inhibiting geranylgeranyl-transferase mimicked the effect of ibandronate on the excess oxidative response. Ibandronate exerted cellular antioxidant effects through the Rac1/NADPH oxidase pathway. These effects may have contributed to the vasoprotective effects on the impaired endothelium in SHRs.

Dimethylarginine dimethylaminohydrolase-1 mediates inhibitory effect of interleukin-10 on angiotensin II-induced hypertensive effects in vascular smooth muscle cells of spontaneously hypertensive rats

In hypertension studies, anti-inflammatory cytokine interleukin-10 (IL-10) has been shown to prevent angiotensin II (Ang II)-induced vasoconstriction and regulate vascular function by down-regulating pro-inflammatory cytokine and superoxide production in vascular cells. However, little is known about the mechanism behind the down-regulatory effect of IL-10 on Ang II-induced hypertensive mediators. In this study, we demonstrated the effects of IL-10 on expression of dimethylarginine dimethylaminohydrolase (DDAH)-1, a regulator of NO bioavailability, as well as the down-regulatory mechanism of action of IL-10 in relation to Ang II-induced hypertensive mediator expression and cell proliferation in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). IL-10 increased DDAH-1 but not DDAH-2 expression and increased DDAH activity. Additionally, IL-10 attenuated Ang II-induced DDAH-1 inhibition in SHR VSMCs. Increased DDAH activity due to IL-10 was mediated mainly through Ang II subtype II receptor (AT2 R) and AMP-activated protein kinase (AMPK) activation. DDAH-1 induced by IL-10 partially mediated the inhibitory action of IL-10 on Ang II-induced 12-lipoxygenase (LO) and endothelin (ET)-1 expression in SHR VSMCs. In addition, the inhibitory effect of IL-10 on proliferation of Ang II-induced VSMCs was mediated partially via DDAH-1 activity. These results suggest that DDAH-1 plays a potentially important role in the anti-hypertensive activity of IL-10 during Ang II-induced hypertension.

Enhanced expression of Giα proteins contributes to the hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats via MAP kinase- and PI3 kinase-independent pathways.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit hyperproliferation, enhanced MAP kinase (MAPK) activity, and overexpression of Giα proteins. This study was undertaken to examine whether the overexpression of Giα proteins contributes to the hyperproliferation of VSMC of SHR through MAPK signaling. The hyperproliferation of VSMC from SHR in the absence and presence of angiotensin II was restored towards those in Wistar-Kyoto (WKY) rats levels by pertussis toxin (PT) treatment. In addition, siRNA knockdown of Giα proteins also resulted in the attenuation of hyperproliferation towards control levels. The overexpression of Giα proteins was also inhibited by MAPK and PI3 kinase (PI3K) inhibitors. In addition, the hyperproliferation and enhanced phosphorylation of ERK1/2 and Akt in VSMC from SHR were attenuated towards WKY levels by the inhibitors of MAPK, PI3K, c-Src, and antioxidants, whereas PT was unable to attenuate the enhanced phosphorylation of ERK1/2 and Akt. Furthermore, 8Br-cAMP and forskolin also attenuated the hyperproliferation of VSMC from SHR. These results suggest that the hyperproliferation of VSMC from SHR may be attributed to the enhanced expression of Giα proteins and increased activation of MAPK and PI3 kinase. However, Giα-mediated hyperproliferation may not be mediated through MAPK- and PI3 kinase-dependent pathways and may involve decreased levels of intracellular cAMP.

CCL5 upregulates IL-10 expression and partially mediates the antihypertensive effects of IL-10 in the vascular smooth muscle cells of spontaneously hypertensive rats.

Interleukin (IL)-10 inhibits angiotensin (Ang) II-induced vascular dysfunction and reduces blood pressure in hypertensive pregnant rats. The chemokine CCL5 has also been shown to downregulate Ang II-induced hypertensive mediators in spontaneously hypertensive rats (SHRs). This study investigated the effects of CCL5 on IL-10 expression, as well as its mechanisms of action in the vascular smooth muscle cells (VSMCs) of SHRs. CCL5 increased IL-10 expression in the VSMCs of SHRs; the s.c. injection of CCL5 (1.5 μg kg(-1), twice a day) for 3 weeks into SHRs with established hypertension upregulated IL-10 expression in both the thoracic aorta and the VSMCs and decreased systolic blood pressure. CCL5-induced the elevation of IL-10 expression, an effect mediated primarily via the activation of an Ang II subtype II receptor (AT2 R). Dimethylarginine dimethylaminohydrolase (DDAH)-1 activity also contributed to the elevation of IL-10 expression via CCL5 in the VSMCs of SHRs. Moreover, CCL5 partially mediated the inhibitory effects of IL-10 on Ang II-induced 12-lipoxygenase (LO) and endothelin (ET)-1 expression in the VSMCs of SHRs. Taken together, this study provides novel evidence that CCL5 plays a role in the upregulation of IL-10 activity in the VSMCs of SHRs.

The Enhancement of Cx45 Expression and Function in Renal Interlobar Artery of Spontaneously Hypertensive Rats at Different Age

Background/Aims: This study was designed to investigate the expression and function of gap junction protein connexin 45 (Cx45) in renal interlobar artery (RIA) of spontaneously hypertensive rats (SHR), and the association between hypertension and enhanced vasoconstrictive response in SHR. Methods: Western blot analysis and pressure myography were used to examine the differences in expression and function of Cx45 in vascular smooth muscle cells (VSMCs) of RIA between SHR and normotensive Wistar-Kyoto (WKY) rats. Results: Our results demonstrated that 1) whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ RIA VSMCs of SHR were significantly greater than those of WKY rats (p<0.05, n=6), suggesting that the coupling of gap junction between VSMCs of RIA was enhanced in SHR; 2) the KCl or phenylephrine (PE)-stimulated RIA constriction was more pronounced in SHR than that in WKY rats (p<0.05, n=10). After applying a gap junction inhibitor 18β-glycyrrhetintic acid (18β-GA), the inhibitory effect of 18β-GA on KCl or PE-induced vasoconstriction was greater in SHR (p<0.05, n=10); and 3) the expression of Cx45 in RIA of SHR was greater than that in WKY rats (p<0.05, n=3) at 4, 12 and 48 wks of age. Conclusions: The hypertension-induced elevation of Cx45 may affect communication between VSMCs and coupling between VSMCs and endothelium, which results in an increased vasoconstrictive response in renal artery and might contribute to the development of hypertension.

Hydrogen sulfide upregulates KATP channel expression in vascular smooth muscle cells of spontaneously hypertensive rats.

The study was designed to investigate whether H2S could upregulate expression of KATP channels in vascular smooth muscle cells (VSMCs), and by this mechanism enhances vasorelaxation in spontaneously hypertensive rats (SHR). Blood pressure, vascular structure, and vasorelaxation were analyzed. Plasma H2S was detected using polarographic sensor. SUR2B and Kir6.1 expressions were detected in VSMCs of SHR and in A7r5 cells as well as primarily cultured ASMCs using real-time PCR, western blot, immunofluorescence, and confocal imaging. Nuclear translocation of forkhead transcription factors FOXO1 and FOXO3a in ASMCs was detected using laser confocal microscopy, and their binding activity with SUR2B and Kir6.1 promoters was examined by chromatin immunoprecipitation. SHR developed hypertension at 18weeks. They showed downregulated vascular SUR2B and Kir6.1 expressions in association with a decreased plasma H2S level. H2S donor, however, could upregulate vascular SUR2B and Kir6.1 expressions, causing a left shift of the vasorelaxation curve to pinacidil and lowered tail artery pressure in the SHR. Also, H2S antagonized endothelin-1 (ET-1)-inhibited KATP expression in A7r5 cells and cultured ASMCs. Mechanistically, H2S inhibited ET-1-stimulated p-FOXO1 and p-FOXO3a expressions (inactivated forms), but increased their nuclear translocation and the ET-1-inhibited binding of FOXO1 and FOXO3a with Kir6.1 and SUR2B promoters in ASMCs. Hence, H2S promotes vasorelaxation of SHR, at least in part, through upregulating the expression of KATP subunits by inhibiting phosphorylation of FOXO1 and FOXO3a, and stimulating FOXO1 and FOXO3a nuclear translocation and their binding activity with SUR2B and Kir6.1 promoters. H2S increased vascular SUR2B and Kir6.1 expression of SHR, promoting vasorelaxation. H2S antagonized ET-1-inhibited KATP expression in A7r5 cells and cultured ASMCs. H2S inhibited ET-1-induced FOXO1 and FOXO3a phosphorylation in ASMCs. H2S promoted FOXO1 and FOXO3a nuclear translocation and binding with target gene promoters.

IL-10 up-regulates CCL5 expression in vascular smooth muscle cells from spontaneously hypertensive rats

An anti-inflammatory cytokine, interleukin-10 (IL-10) exerts inhibitory effects on vascular inflammation. Chemokines promote vascular inflammation and play a pathogenic role in the development and maintenance of hypertension. However, chemokine CCL5 has down-regulatory effects on angiotensin II (Ang II)-induced hypertensive mediators. In the present study, IL-10 increased CCL5 expression and attenuated Ang II-induced CCL5 inhibition significantly in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR), whereas TGF-β had no effect on CCL5 expression or Ang II-induced CCL5 inhibition. Increased CCL5 expression due to IL-10 was mediated mainly through AT2 R activation. Additionally, IL-10 increased activation of AMP-activated protein kinase (AMPK), which further mediated the up-regulatory effect of IL-10 on CCL5 expression. Attenuation of Ang II-induced CCL5 inhibition by IL-10 was associated with suppression of NF-кB activation, and IL-10 inhibited both Ang II-induced IкB-α and IкB-β degradation in SHR VSMCs. Moreover, IL-10 partially mediated the inhibitory effects of CCL5 on Ang II-induced 12-lipoxygenase (LO) and endothelin (ET)-1 expression in SHR VSMCs. Taken together, this study provides novel evidence that IL-10 plays an up-regulatory role in the anti-hypertensive activity of CCL5 in SHR VSMCs.

Role of gap junctions in the contractile response to agonists in the mesenteric artery of spontaneously hypertensive rats

To investigate the effects of hypertension on the changes in gap junctions between vascular smooth muscle cells (VSMCs) in the mesenteric artery (MA) of spontaneously hypertensive rats (SHRs). Whole-cell patch clamp, pressure myography, real-time quantitative reverse transcription PCR (qRT-PCR), western blot analysis and transmission electron microscopy were used to examine the differences in expression and function of the gap junction between MA VSMCs of SHR and control normotensive Wistar-Kyoto (WKY) rats. (1) Whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ MA VSMCs of SHR were significantly greater than those of WKY rats (P<0.05), suggesting enhanced gap junction coupling between MA VSMCs of SHR. (2) The administration of phenylephrine (PE) and KCl (an endothelium-independent vasoconstrictor) initiated more pronounced vasoconstriction in SHR versus WKY rats (P<0.05). Furthermore, 2-APB (a gap junction inhibitor) attenuated PE- and KCl-induced vasoconstriction, and the inhibitory effects of 2-APB were significantly greater in SHR (P<0.05). (3) The expression of connexin 45 (Cx45) mRNA and protein in the MA was greater in SHR versus WKY rats (P<0.05). The level of phosphorylated Cx43 was significantly higher in SHR versus WKY rats (P<0.05), although the expression of total Cx43 mRNA and protein in the MA was equivalent between SHR and WKY rats. Electron microscopy revealed that the gap junctions were significantly larger in SHR versus WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may contribute to the enhancement of communication across gap junctions between MA VSMCs of SHR, which may increase the contractile response to agonists.

Effect of CCL5 on dimethylarginine dimethylaminohydrolase-1 production in vascular smooth muscle cells from spontaneously hypertensive rats

Chemokines promote vascular inflammation and play a pathogenic role in the development and maintenance of hypertension. However, in our previous study, chemokine CCL5 was shown to reduce Ang II-induced 12-lipoxygenase (12-LO) production as well as proliferation in vascular smooth muscle cells (VSMCs) obtained from spontaneously hypertensive rats (SHR). Dimethylarginine dimethylaminohydrolase (DDAH) acts as an important regulator of vascular function by metabolizing and regulating plasma asymmetric (NG,NG) dimethylarginine (ADMA), a major risk factor for cardiovascular disease. Therefore, in this study, we investigated the effect of CCL5 on DDAH-1 production in SHR VSMCs. Constitutive expression of DDAH-1 in VSMCs from SHR was higher than that in VSMCs from normotensive Wistar Kyoto rats (WKY), whereas expression of DDAH-2 was not significantly different between SHR and WKY VSMCs. CCL5 increased DDAH-1 production and attenuated Ang II-induced DDAH-1 inhibition in SHR VSMCs. In addition, although CCL5 did not affect the level of asymmetric (NG,NG) dimethylarginine (ADMA), it attenuated Ang II-induced ADMA production through DDAH-1 activity. DDAH-1 induction by CCL5 was mediated by the Ang II subtype 2 receptor (AT2 R) pathway. Further, attenuation of Ang II-induced 12-LO and endothelin-1 (ET-1) expression by CCL5 could be attributed to DDAH-1 activity. These findings combined with our previous results suggest that CCL5 is a potential down-regulatory factor in Ang II-induced vascular hypertension.

Hsp70 regulation on Nox4/p22phox and cytoskeletal integrity as an effect of losartan in vascular smooth muscle cells

A series of signaling cascades are activated after angiotensin II binds to angiotensin II type I receptor (AT1R), a peptide that is an important mediator of oxidative stress. Hsp70 regulates a diverse set of signaling pathways through interactions with proteins. Here, we tested the hypothesis of angiotensin II AT1R inhibition effect on Hsp70 interaction with Nox4/p22phox complex and Hsp70 leading to actin cytoskeleton modulation in spontaneously hypertensive rats (SHR) vascular smooth muscle cells (VSMCs). SHR and Wistar-Kyotto rats (VSMCs from 8 to 10weeks) were stimulated with angiotensin II (100nmol/L) for 15min (AII), treated with losartan (100nmol/L) for 90min (L), and with losartan for 90min plus angiotensin in the last 15min (L + AII). Whereas SHR VSMCs exposure to angiotensin II overexpressed AT1R and Nox4 nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase and slightly downregulated caveolin-1 expression, losartan decreased AT1R protein levels and increased caveolin-1 and Hsp70 expression in SHR VSMC membranes. Immunoprecipitation and immunofluorescence confocal microscopy proved interaction and colocalization of membrane translocated Hsp70 and Nox4/p22phox. Increased levels of Hsp70 contrast with the decreased immunoprecipitation of Nox4/p22phox and RhoA in membranes from SHR VSMCs (L) vs SHR VSMCs (AII). Hsp72 depletion resulted in higher Nox4 expression and increased NADPH oxidase activity in VSMCs (L + AII) from SHR when contrasted with nontransfected VSMCs (L + AII). After Hsp72 knockdown in SHR VSMCs, losartan could not impair angiotensin II-enhanced stress fiber formation and focal adhesion assembly. In conclusion, our data showing a negative regulation of Hsp70 on Nox4/p22phox demonstrates a possible mechanism in explaining the antioxidative function joined to cytoskeletal integrity modulation within the effects of losartan in VSMCs from SHR.

Insulin Like Growth Factor Binding Protein-5 Regulates Excessive Vascular Smooth Muscle Cell Proliferation in Spontaneously Hypertensive Rats via ERK 1/2 Phosphorylation

Insulin-like growth factor binding proteins (IGFBPs) are important components of insulin growth factor (IGF) signaling pathways. One of the binding proteins, IGFBP-5, enhances the actions of IGF-1, which include the enhanced proliferation of smooth muscle cells. In the present study, we examined the expression and the biological effects of IGFBP-5 in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). The levels of IGFBP-5 mRNA and protein were found to be higher in the VSMC from SHR than in those from WKY. Treatment with recombinant IGFBP-5-stimulated VSMC proliferation in WKY to the levels observed in SHR. In the VSMCs of WKY, incubation with angiotensin (Ang) II or IGF-1 dose dependently increased IGFBP-5 protein levels. Transfection with IGFBP-5 siRNA reduced VSMC proliferation in SHR to the levels exhibited in WKY. In addition, recombinant IGFBP-5 significantly up-regulated ERK1/2 phosphorylation in the VSMCs of WKY as much as those of SHR. Concurrent treatment with the MEK1/2 inhibitors, PD98059 or U0126 completely inhibited recombinant IGFBP-5-induced VSMC proliferation in WKY, while concurrent treatment with the phosphatidylinositol-3 kinase inhibitor, LY294002, had no effect. Furthermore, knockdown with IGFBP-5 siRNA inhibited ERK1/2 phosphorylation in VSMC of SHR. These results suggest that IGFBP-5 plays a role in the regulation of VSMC proliferation via ERK1/2 MAPK signaling in hypertensive rats.

Comparison of 12-lipoxygenase expression in vascular smooth muscle cells from old normotensive Wistar-Kyoto rats with spontaneously hypertensive rats

Vascular aging and essential hypertension cause similar structural and molecular modifications in the vasculature. The 12-lipoxygenase (LO) pathway of arachidonic acid metabolism is linked to cell growth and the pathology of hypertension. Thus, elevated expression of 12-LO has been observed in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). In the present study, we investigated the differences in 12-LO expression and activity between VSMCs from old normotensive Wistar-Kyoto rats (old WKY, 90-week old) and SHR (13-week old). The protein and mRNA expression of basal or angiotensin II (Ang II)-induced 12-LO in old WKY VSMCs were higher than those in SHR VSMCs. The degradation rate of 12-LO mRNA in old WKY VSMCs was slower than that in SHR VSMCs. However, basal or Ang II-induced 12-LO mRNAs in both old WKY and SHR VSMCs decayed more rapidly than that in young WKY (13-week old) VSMCs. Higher expression of 12-LO in old WKY VSMCs than in SHR VSMCs was correlated with the expression level of Ang II subtype 1 receptor (AT(1)R). The reduced levels of nitric oxide (NO) in old WKY and SHR VSMCs compared with young WKY VSMCs were similar, and there was no significant difference in NO production between old WKY and SHR VSMCs transfected with 12-LO siRNA. In addition, in contrast to the proliferation of SHR VSMCs, the proliferation of old WKY VSMCs was not dependent on 12-LO activation. These results suggest that the potential role of 12-LO in normotensive aging vasculature may be different from that in SHR vasculature.