Vascular Smooth Muscle Cell Layer Research Articles

Abstract 16749: Influence of Dual-Specificity Protein Phosphatase 5 on Structural and Mechanical Properties of Rat Middle Cerebral Artery

Dual-specificity protein phosphatase 5 (DUSP5) is a multifunctional phosphatase that modulates signaling cascades by catalyzing the dephosphorylation of both threonine and tyrosine residues. We recently reported that KO of Dusp5 enhances cerebral and renal hemodynamics and cognitive function. This effect is associated with increased pPKC and pERK1/2 levels in arteries and arterioles of the brain and kidneys. Further, we found that KO of Dusp5 contributes to the regulation of arteriolar passive mechanical properties with higher myogenic tones, better distensibility, greater compliance, and less stiffness. The present study focuses on the structural and mechanical properties of rat middle cerebral artery (MCA) to evaluate whether it contributes to enhanced vascular and cognitive function in Dusp5 KO rats. Vascular smooth muscle cell layer numbers and collagen content in the wall of the MCA are similar in Dusp5 KO and control rats. While external elastic lamina was absent in the MCA in both strains, internal elastic lamina was thicker in the MCA of Dusp5 KO vs. control rats (2.33 ± 0.02 vs. 1.85 ± 0.03 μm) and associated with higher autofluorescence intensity (953.76 ± 10.03 vs. 737.31 ± 64.88 au), smaller fenestrae areas (51.31 ± 3.62 vs. 70.46 ± 2.24), and fewer number of fenestrations (138.19 ± 8.86 vs. 374.73 ± 17.02 μm 2 ). Even though the myogenic tone is enhanced in Dusp5 KO vs. control rats (52.37 ± 1.65 vs. 44.87 ± 2.00%), other passive mechanical properties (wall thickness, cross-sectional area, wall-to-lumen ratio, wall tension, distensibility, incremental distensibility, circumferential wall strain, circumferential wall stress, and elastin modulus) of the MCA of Dusp5 KO rats do not exhibit significant changes. The findings suggest that while Dusp5 KO has a limited impact on changing the structural and mechanical properties of the MCA, its role in enhancing hemodynamics and cognitive function is primarily attributed to its enzymatic function.

Abstract P3125: Cldn11 Is A Candidate For Vascular Smooth Muscle Cell Plasticity Regulator In Atherosclerosis Pathology

Claudin-11(CLDN11) is a component of tight junction, and important for normal CNS functions. The function of CLDN11 is well elucidated in oligodendrocytes and CNS system, but its expression pattern according to human vascular bed such as intracranial and extracranial, role in the vascular smooth muscles (vSMCs) plasticity and atherosclerosis is still unknown. Human left anterior descending coronary artery (LAD) and middle cerebral artery (MCA) vascular smooth muscle layer was dissected from the dead body. CLDN11 expression is identified in LAD and MCA vascular smooth muscle cell layer, and the CLDN11 expression is higher in MCA than in LAD (p<0.01). Interestingly, the CLDN11 expression is decreased in vSMCs around the atherosclerotic lesions compare to non-atheromatous lesions by immunohistochemistry. To determine the functional role of CLDN11 in vSMCs, microarray analysis was performed following siCLDN11 knockdown. Compared to normal HASMC (human aortic vascular smooth muscle cells), siCLDN11-HASMC was showed increased gene expression such as RSPO3, CXCL8, EGR3, CYP1B1, TNFAIP2, PTGS2, HEY1, BMPER and these set was related to cell migration, proliferation and angiogenesis. CLDN11 knockdown vSMCs showed increased the cell migration of HASMC measured by wound healing, tube formation, and cell cycle assay, and reduced cas-3 cleavage. Finally, CLDN11 expression was inversely correlated with the progression of coronary atherosclerosis and the sudden cardiac death with complicated coronary atherosclerosis. This study shows that CLDN11 play a role in the initiation and propagation of atherosclerosis by regulating the plasticity of vSMCs, suggesting the possibility of biological marker for atherosclerosis in clinical diagnosis (This work was supported by NRF-2019R1A2C1089108, NRF-2019M3E5D1A02068082, COMPA-2022C100, and BK21 Program, Center for Glocal Future Biomedical Scientists at Chonnam National University).

Nitrate-Functionalized poly(ε-Caprolactone) Small-Diameter Vascular Grafts Enhance Vascular Regeneration via Sustained Release of Nitric Oxide.

Artificial small-diameter vascular grafts (SDVG) fabricated from synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL), exhibit beneficial mechanical properties but are often faced with issues impacting their long-term graft success. Nitric oxide (NO) is an important physiological gasotransmitter with multiple roles in orchestrating vascular tissue function and regeneration. We fabricated a functional vascular graft by electrospinning of nitrate-functionalized poly(ε-caprolactone) that could release NO in a sustained manner via stepwise biotransformation in vivo. Nitrate-functionalized SDVG (PCL/NO) maintained patency following abdominal arterial replacement in rats. PCL/NO promoted cell infiltration at 3-months post-transplantation. In contrast, unmodified PCL SDVG showed slow cell in-growth and increased incidence of neointima formation. PCL/NO demonstrated improved endothelial cell (EC) alignment and luminal coverage, and more defined vascular smooth muscle cell (VSMC) layer, compared to unmodified PCL SDVG. In addition, release of NO stimulated Sca-1+ vascular progenitor cells (VPCs) to differentiate and contribute to rapid luminal endothelialization. Furthermore, PCL/NO inhibited the differentiation of VPCs into osteopontin-positive cells, thereby preventing vascular calcification. Overall, PCL/NO demonstrated enhanced cell ingrowth, EC monolayer formation and VSMC layer regeneration; whilst inhibiting calcified plaque formation. Our results suggested that PCL/NO could serve as promising candidates for improved and long-term success of SDVG implants.

Procedures to Evaluate the Role of Heparan Sulfate on the Reactivity of Resistance and Conductance Arteries Ex Vivo.

Evidence is emerging that disruption of the endothelial glycocalyx might contribute importantly to arterial dysfunction in the context of diabetes. One approach to assess the integrity of the endothelium and the vascular smooth muscle cell layer, in the absence of neural, humoral, and mechanical influences, is by measuring arterial vasomotion ex vivo. Here we describe a procedure to assess non-receptor-mediated vasoconstriction, receptor-mediated vasoconstriction, and endothelium-dependent and -independent vasodilation, in resistance and conductance arteries pressurized to 60mmHg. In addition to evaluating vasoreactivity using isobaric approaches, the same experimental set-up can be used to initiate a pressure gradient across the artery such that intraluminal, flow-mediated vasodilation can be measured. After recording endothelium-dependent vasodilation using isobaric or flow-mediated approaches, identical interventions can be completed in the presence of enzymes that cleave biologically active heparan sulfates into inactive disaccharide and oligosaccharide fragments to assess the contribution from: (a) endothelial-derived substances (e.g., nitric oxide via nitric oxide synthase inhibition); or (b) important components of the glycocalyx (e.g., removal of heparan sulfate via heparitinase III treatment). Here, we show that acute disruption of a predominant glycosaminoglycan i.e., heparan sulfate impairs intraluminal flow-mediated vasodilation in murine resistance arteries.

Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells.

The nuclear factor of activated T-cells 5 (NFAT5) is a transcriptional regulator of macrophage activation and T-cell development, which controls stabilizing responses of cells to hypertonic and biomechanical stress. In this study, we detected NFAT5 in the media layer of arteries adjacent to human arteriosclerotic plaques and analyzed its role in vascular smooth muscle cells (VSMCs) known to contribute to arteriosclerosis through the uptake of lipids and transformation into foam cells. Exposure of both human and mouse VSMCs to cholesterol stimulated the nuclear translocation of NFAT5 and increased the expression of the ATP-binding cassette transporter Abca1, required to regulate cholesterol efflux from cells. Loss of Nfat5 promoted cholesterol accumulation in these cells and inhibited the expression of genes involved in the management of oxidative stress or lipid handling, such as Sod1, Plin2, Fabp3, and Ppard. The functional relevance of these observations was subsequently investigated in mice fed a high-fat diet upon induction of a smooth muscle cell-specific genetic ablation of Nfat5 (Nfat5(SMC)-/- ). Under these conditions, Nfat5(SMC)-/- but not Nfat5fl/fl mice developed small, focal lipid-rich lesions in the aorta after 14 and 25weeks, which were formed by intracellular lipid droplets deposited in the sub-intimal VSMCs layer. While known for being activated by external stimuli, NFAT5 was found to mediate the expression of VSMC genes associated with the handling of lipids in response to a cholesterol-rich environment. Failure of this protective function may promote the formation of lipid-laden arterial VSMCs and pro-atherogenic vascular responses.

Development of Multi-layer Tubular Vascular Scaffold to Enhance Compliance by Exhibiting a Negative Poisson’s Ratio

Synthetic small diameter vascular grafts frequently fail owing to intimal hyperplasia, results from mismatched compliance between the vascular graft and native vessels. A vascular graft with a negative Poisson's ratio (NPR, materials expand transversely when pulled axially) was suggested to enhance compliance. We produced a three-layer tubular vascular scaffold with NRP properties. The luminal side consisted of nanosized electrospun fibers for endothelial cell (EC) growth. The middle layer was an NPR structure created using 3D printing, and the outer layer was a microsized electrospun fiber layer for vascular smooth muscle cell (VSMC) growth. The developed multi-layer tubular vascular scaffold contained NPR value. And the NPR vascular scaffold showed 1.7 time higher compliance than the PPR scaffold and 3.8 times higher than that of commercial polytetrafluoroethylene (PTFE) vascular graft. In addition, the ECs and VSMCs were well survived and proliferated on the scaffold during 10 days of culture. From the optimized co-culture condition of the VSMCs and ECs that VSMC phenotype changed was inhibited, we successfully generated a thin luminal layer, which consisted of ECs and the proper thickness of the VSMC layer under the ECs. This scaffold may have a potential to replace conventional artificial vascular graft by providing enhanced compliance and improved cell culture environment.

BMP9 and BMP10 Act Directly on Vascular Smooth Muscle Cells for Generation and Maintenance of the Contractile State.

Vascular smooth muscle cells (VSMCs) show a remarkable phenotypic plasticity, allowing acquisition of contractile or synthetic states, but critical information is missing about the physiologic signals, promoting formation, and maintenance of contractile VSMCs in vivo. BMP9 and BMP10 (bone morphogenetic protein) are known to regulate endothelial quiescence after secretion from the liver and right atrium, whereas a direct role in the regulation of VSMCs was not investigated. We studied the role of BMP9 and BMP10 for controlling formation of contractile VSMCs. We generated several cell type-specific loss- and gain-of-function transgenic mouse models to investigate the physiologic role of BMP9, BMP10, ALK1 (activin receptor-like kinase 1), and SMAD7 in vivo. Morphometric assessments, expression analysis, blood pressure measurements, and single molecule fluorescence in situ hybridization were performed together with analysis of isolated pulmonary VSMCs to unravel phenotypic and transcriptomic changes in response to absence or presence of BMP9 and BMP10. Concomitant genetic inactivation of Bmp9 in the germ line and Bmp10 in the right atrium led to dramatic changes in vascular tone and diminution of the VSMC layer with attenuated contractility and decreased systemic as well as right ventricular systolic pressure. On the contrary, overexpression of Bmp10 in endothelial cells of adult mice dramatically enhanced formation of contractile VSMCs and increased systemic blood pressure as well as right ventricular systolic pressure. Likewise, BMP9/10 treatment induced an ALK1-dependent phenotypic switch from synthetic to contractile in pulmonary VSMCs. Smooth muscle cell-specific overexpression of Smad7 completely suppressed differentiation and proliferation of VSMCs and reiterated defects observed in adult Bmp9/10 double mutants. Deletion of Alk1 in VSMCs recapitulated the Bmp9/10 phenotype in pulmonary but not in aortic and coronary arteries. Bulk expression analysis and single molecule RNA-fluorescence in situ hybridization uncovered vessel bed-specific, heterogeneous expression of BMP type 1 receptors, explaining phenotypic differences in different Alk1 mutant vessel beds. Our study demonstrates that BMP9 and BMP10 act directly on VSMCs for induction and maintenance of their contractile state. The effects of BMP9/10 in VSMCs are mediated by different combinations of BMP type 1 receptors in a vessel bed-specific manner, offering new opportunities to manipulate blood pressure in the pulmonary circulation.

VSMC-Specific Deletion of FAM3A Attenuated Ang II-Promoted Hypertension and Cardiovascular Hypertrophy.

Dysregulated purinergic signaling transduction plays important roles in the pathogenesis of cardiovascular diseases. However, the role and mechanism of vascular smooth muscle cell (VSMC)-released ATP in the regulation of blood pressure, and the pathogenesis of hypertension remain unknown. FAM3A (family with sequence similarity 3 member A) is a new mitochondrial protein that enhances ATP production and release. High expression of FAM3A in VSMC suggests it may play a role in regulating vascular constriction and blood pressure. To determine the role and mechanism of FAM3A-ATP signaling pathway in VSMCs in the regulation of blood pressure and the pathogenesis of hypertension. In the media layer of hypertensive rat and mouse arteries, and the internal mammary artery of hypertensive patients, FAM3A expression was increased. VSMC-specific deletion of FAM3A reduced vessel contractility and blood pressure levels in mice. Moreover, deletion of FAM3A in VSMC attenuated Ang II (angiotensin II)-induced vascular constriction and remodeling, hypertension, and cardiac hypertrophy in mice. In cultured VSMCs, Ang II activated HSF1 (heat shock factor 1) to stimulate FAM3A expression, activating ATP-P2 receptor pathway to promote the change of VSMCs from contractile phenotype to proliferative phenotype. In the VSMC layer of spontaneously hypertensive rat arteries, Ang II-induced hypertensive mouse arteries and the internal mammary artery of hypertensive patients, HSF1 expression was increased. Treatment with HSF1 inhibitor reduced artery contractility and ameliorated hypertension of spontaneously hypertensive rats. FAM3A is an important regulator of vascular constriction and blood pressure. Overactivation of HSF1-FAM3A-ATP signaling cascade in VSMCs plays important roles in Ang II-induced hypertension and cardiovascular diseases. Inhibitors of HSF1 could be potentially used to treat hypertension.

Uremic Vascular Calcification Is Correlated With Oxidative Elastic Lamina Injury, Contractile Smooth Muscle Cell Loss, Osteogenesis, and Apoptosis: The Human Pathobiological Evidence.

Background: Uremic vascular calcification (UVC) is reminiscent of osteogenesis and apoptosis in vascular smooth muscle cell (VSMC). We aimed to identify how circulating procalcific particles dramatically leak into VSMC layer in human tissue models of vascular rings.Methods: According to baseline estimated glomerular filtration rate (eGFR), patients following lower extremity amputation were divided into three groups: normal renal function (eGFR ≧ 60 ml/min), mild-to-moderate (15 ml/min < eGFR ≧ 60 ml/min) and severe chronic kidney disease (CKD) (eGFR ≦ 15 ml/min). Arterial specimens with immunohistochemistry stain were quantitatively analyzed for UVC, internal elastic lamina (EL) disruption, α-SMA, osteogenesis, apoptosis, and oxidative injury. Correlations among UVC severity, eGFR, EL disruption, osteogenesis, and oxidative injury were investigated.Results: CKD arteries were associated with eGFR-dependent EL disruption corresponding to UVC severity. CKD arteries exhibited lower α-SMA, higher expressions of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), indicative of contractile VSMC loss, and apoptosis. Enhanced expressions of alkaline phosphatase and Runx2 were presented in VSMCs of CKD arteries, indicative of osteogenic differentiation. Above eGFR-dependent UVC and EL disruption correlated expressions of 8-hydroxy-2′-deoxyguanosine (8-OHdG), indicating oxidative EL injury promoted procalcific processes.Conclusions: Circulating uremic milieu triggers vascular oxidative stress, leading to progressive internal EL disruption as a key event in disabling VSMC defense mechanisms and catastrophic mineral ion influx into VSMC layer. Oxidative EL injury begins in early CKD, corresponding with active VSMC re-programming, apoptosis, and ultimately irremediable UVC. In light of this, therapeutic strategies targeting oxidative tissue injury might be of vital importance to hinder the progression of UVC related cardiovascular events.

Orthogonal co-cultivation of smooth muscle cell and endothelial cell layers to construct in vivo-like vasculature.

Development of a three-dimensional (3D) vascular co-cultivation system is one of the major challenges to provide an advanced analytical platform for studying blood vessel related diseases. To date, however, the in vivo-like vessel system has not been fully realized due to the difficulty of co-cultivation of the cells with orthogonal alignment. In this study, we report the utilization of microfabrication technology to construct biomimetic 3D co-cultured vasculature. First, microwrinkle patterns whose direction was perpendicular to the axis of a circular microfluidic channel were fabricated, and vascular smooth muscle cells (VSMCs) were cultured inside the microchannel, leading to an in vivo-like circumferential VSMC layer. Then, human umbilical vein endothelial cells (HUVECs) were co-cultured on the circumferentially aligned VSMC, and the success of double layer formation of HUVEC-VSMC in the circular microchannel could be monitored. After HUVEC cultivation, we applied shear flow in order to induce the orientation of HUVEC parallel to the axis, and the analysis of orientation angle and spreading area of HUVECs indicated that they were changed by shear stress to be aligned to the direction of flow. Thus, the HUVEC and VSMC layer could be aligned with a distinct direction. The expression level of VE-Cadherin located at the boundary of HUVECs implies in vivo-like vascular behavior. The proposed in vitro microfluidic vascular assay platform would be valuable for studying vascular diseases with high reliability due to in vivo-likeness.

Vasomotor Impact of Perivascular Adipose Tissue on Coronary Circulation

Research during the past decade has highlighted the functional role of perivascular adipose tissue (PVAT) in regulating the contractility of the underlying vascular smooth muscle cell layer. However, the mechanisms underlying these observations are not fully understood. PVAT has a protective role as it could induce vasorelaxation in different anatomical positions. However, there is a considerable evidence that the development and progression of coronary artery disease could be attributed to coronary PVAT. This review provides a comprehensive review of the vasomotor effect of PVAT that identifies the coronary PVAT as a necessary paracrine organ with a significant role in physiology and associated pathologies, and calls for basic cardiovascular research to solve this issue.

Downregulation of the α1- and β1-subunit of sGC in Arterial Smooth Muscle Cells of OPSCC Is HPV-Independent

The nitric oxide (NO)–sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an α and β subunit. NO binds to heme of the β1-subunit of sGC, activates the enzyme in the reduced heme iron state in vascular smooth muscle cells (VSMCs), and generates cGMP-inducing vasodilatation and suppression of VSMC proliferation. In the complex tumor milieu with higher levels of reactive oxygen species (ROS), sGC heme iron may become oxidized and insensitive to NO. To change sGC from an NO-insensitive to NO-sensitive state or NO-independent manner, protein expression of sGC in VSMC is required. Whether sGCα1β1 exists at the protein level in arterial VSMCs of oropharyngeal squamous cell carcinoma (OPSCC) is unknown. In addition, whether differences in the genetic profile between human papillomavirus (HPV)–positive and HPV-negative OPSCC contributes to the regulation of sGCα1β1 is unclear. Therefore, we compared the effects of HPV-positive and HPV-negative OPSCC on the expression of sGCα1β1 in arterial VSMCs from tumor-free and tumor-containing regions of human tissue sections using quantitative immunohistochemistry. In comparison to the tumor-free region, we found a decrease in expression of both α1- and β1-subunits in the arterial VSMC layer of the tumor-containing areas. The OPSCC-induced significant downregulation of the α1- and β1-subunits of sGC in arterial VSMC was HPV-independent. We conclude that the response of sGC to NO in tumor arterial VSMCs may be impaired by oxidation of the heme of the β1-subunit, and thus, α1- and β1-subunits of sGC could be targeted to degradation under oxidative stress in OPSCC in an HPV-independent manner. The degradation of sGCα1β1 in VSMCs may result in increased proliferation of VSMCs, promoting tumor arteriogenesis in OPSCC. This can be interrupted by preserving the active heterodimer sGCα1β1 in arterial VSMCs.

Haptoglobin Preserves Vascular Nitric Oxide Signaling during Hemolysis.

Hemolysis occurs not only in conditions such as sickle cell disease and malaria but also during transfusion of stored blood, extracorporeal circulation, and sepsis. Cell-free Hb depletes nitric oxide (NO) in the vasculature, causing vasoconstriction and eventually cardiovascular complications. We hypothesize that Hb-binding proteins may preserve vascular NO signaling during hemolysis. Characterization of an archetypical function by which Hb scavenger proteins could preserve NO signaling during hemolysis. We investigated NO reaction kinetics, effects on arterial NO signaling, and tissue distribution of cell-free Hb and its scavenger protein complexes. Extravascular translocation of cell-free Hb into interstitial spaces, including the vascular smooth muscle cell layer of rat and pig coronary arteries, promotes vascular NO resistance. This critical disease process is blocked by haptoglobin. Haptoglobin does not change NO dioxygenation rates of Hb; rather, the large size of the Hb:haptoglobin complex prevents Hb extravasation, which uncouples NO/Hb interaction and vasoconstriction. Size-selective compartmentalization of Hb functions as a substitute for red blood cells after hemolysis and preserves NO signaling in the vasculature. We found that evolutionarily and structurally unrelated Hb-binding proteins, such as PIT54 found in avian species, functionally converged with haptoglobin to protect NO signaling by sequestering cell-free Hb in large protein complexes. Sequential compartmentalization of Hb by erythrocytes and scavenger protein complexes is an archetypical mechanism, which may have supported coevolution of hemolysis and normal vascular function. Therapeutic supplementation of Hb scavengers may restore vascular NO signaling and attenuate disease complications in patients with hemolysis.

Porcelain Aorta

Calcification of the thoracic aorta is often associated with valvular and coronary calcification, reflecting an underlying atherosclerotic process.1,2 It has been found to be associated with an increased rate of mortality and cardiovascular disease.1 Porcelain aorta (PA) is extensive calcification of the ascending aorta or aortic arch that can be completely or near completely circumferential.3–5 This entity is rare in the general population, but it has an increasing incidence in older patients and in patients with coronary artery disease (CAD) or aortic stenosis (AS).6,7 The clinical relevance is based on the fact that it can complicate surgical aortic valve replacement (SAVR) for the treatment of severe AS by preventing safe access via the ascending aorta. PA is associated with increased morbidity and mortality, especially as a result of increased perioperative stroke risk.8,9 Recently, transcatheter aortic valve replacement (TAVR) has emerged as a less invasive and feasible treatment option in patients at high risk for conventional SAVR.10 In some series, ≈20% (5%–33%) of patients undergoing TAVR were diagnosed with PA.11 Inconsistencies in the definition and the use of different diagnostic modalities contribute to this wide range of PA prevalence. We reviewed the available published data to seek a consistent, clinically relevant definition based on contemporary imaging, a firm understanding of the pathogenesis and associations, and the clinical implications of this disease entity. PA has been used to address extensive circumferential or nearly circumferential calcification of the thoracic aorta such that it precludes safe cross-clamping or entry to the ascending aorta. However, there is no clear description or definition used; thus, cardiac surgeons and cardiologists use this term inconsistently. In the 1980s, Coselli and Crawford12 initially described 2 …

Expression and significance of NELIN and SM22α in varicose vein tissue.

The aim of the present study was to investigate the expression of NELIN and SM22α in lower extremity varicose vein tissue, and their association with varicose veins. Tissue samples were collected from 18 patients with lower extremity varicose veins for the experimental group, while normal great saphenous vein tissue was reserved during coronary artery bypass surgery from 14 patients for the controls. Reverse transcription polymerase chain reaction (RT-PCR) analysis was applied to detect the mRNA expression levels of NELIN and SM22α, while immunohistochemical techniques were used to detect the protein expression levels in the normal and abnormal veins. RT-PCR results revealed that the mRNA expression levels of NELIN and SM22α in the experimental group decreased significantly when compared with the control group (P<0.01). In the two groups, immunohistochemical staining demonstrated that NELIN and SM22α were primarily expressed in the cytoplasm of smooth muscle cells, and the expression quantity decreased significantly in the experimental group when compared with the control group (P<0.05). The low expression of SM22α in the primary lower limb varicose vein tissue indicated that the vascular smooth muscle cell layer had transformed from a contractile to a secretory phenotype, which may have resulted in the remodeling of the vein walls and the occurrence of varicose veins. Therefore, NELIN and SM22α were demonstrated to play a key role in the development of varicosity.

Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).

In human genetic studies a single nucleotide polymorphism within the salt-inducible kinase 1 (SIK1) gene was associated with hypertension. Lower SIK1 activity in vascular smooth muscle cells (VSMCs) leads to decreased sodium-potassium ATPase activity, which associates with increased vascular tone. Also, SIK1 participates in a negative feedback mechanism on the transforming growth factor-β1 signaling and downregulation of SIK1 induces the expression of extracellular matrix remodeling genes. To evaluate whether reduced expression/activity of SIK1 alone or in combination with elevated salt intake could modify the structure and function of the vasculature, leading to higher blood pressure. SIK1 knockout (sik1(-/-)) and wild-type (sik1(+/+)) mice were challenged to a normal- or chronic high-salt intake (1% NaCl). Under normal-salt conditions, the sik1(-/-) mice showed increased collagen deposition in the aorta but similar blood pressure compared with the sik1(+/+) mice. During high-salt intake, the sik1(+/+) mice exhibited an increase in SIK1 expression in the VSMCs layer of the aorta, whereas the sik1(-/-) mice exhibited upregulated transforming growth factor-β1 signaling and increased expression of endothelin-1 and genes involved in VSMC contraction, higher systolic blood pressure, and signs of cardiac hypertrophy. In vitro knockdown of SIK1 induced upregulation of collagen in aortic adventitial fibroblasts and enhanced the expression of contractile markers and of endothelin-1 in VSMCs. Vascular SIK1 activation might represent a novel mechanism involved in the prevention of high blood pressure development triggered by high-salt intake through the modulation of the contractile phenotype of VSMCs via transforming growth factor-β1-signaling inhibition.

Calcium Sensing Receptor Regulating Smooth Muscle Cells Proliferation Through Initiating Cystathionine-Gamma-Lyase/Hydrogen Sulfide Pathway in Diabetic Rat

Aims: Hydrogen sulfide (H₂S) inhibits the proliferation of vascular smooth muscle cells (VSMCs). However, how cystathionine-gamma-lyase (CSE), a major enzyme that produces H₂S, is regulated remains unknown. Whether calcium-sensing receptor (CaSR) inhibits the proliferation of VSMCs by regulating the endogenous CSE/H₂S pathway in diabetic rat has not been previously investigated. Methods and Results: The morphological and ultrastructure alterations were tested by transmission electron microscopy, changes in the H₂S concentration and the relaxation of the mesenteric secondary artery loop of diabetic rats were determined by Multiskan spectrum microplate spectrophotometer and isometric force transducer. Additionally, the expression levels of CaSR, CSE and Cyclin D1 in the mesenteric arteries of rats were examined by western blotting. The intracellular calcium concentration, the expression of p-CaMK II (phospho-calmodulin kinases II), CSE activity, the concentration of endogenous H₂S and the proliferation of cultured VSMCs from rat thoracic aortas were measured by using confocal microscope, western blotting, microplate spectrophotometer, MTT and BrdU, respectively. The VSMC layer thickened, the H₂S concentration dropped, the relaxation of the mesenteric secondary artery rings weakened, and the expression of CaSR and CSE decreased whereas the expression of Cyclin D1 increased in diabetic rats compared with the control group. The [Ca²⁺]_i of VSMCs increased upon treatment with CaSR agonists (10 µM Calindol and 2.5 mM CaCl₂), while it decreased upon administration of calhex231, U73122 and 2-APB. The expression of p-CaMK II and CSE increased upon treatment with CaSR agonists in VSMCs. CSE activity and the endogenous H₂S concentration decreased in response to high glucose, while it increased with treatment of CaSR agonists. The proliferation rate increased in response to high glucose, and CaSR agonists or NaHS significantly reversed the proliferation of VSMCs caused by high glucose. Conclusions: Our results demonstrated that CaSR regulated the endogenous CSE/H₂S pathway to inhibit the proliferation of VSMCs in both diabetic and high glucose models.

In This Issue…

HomeCirculation ResearchVol. 113, No. 8In This Issue… Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue… Originally published27 Sep 2013https://doi.org/10.1161/01.res.0000435046.80748.fdCirculation Research. 2013;113:945Smad2 in VSMC Differentiation of Neural Crest (p e76)Smad2 and MRTFB cooperate to turn neural crest cells into vascular smooth muscle, report Xie et al.Download figureDownload PowerPointProper differentiation of vascular smooth muscle cells (VSMCs) is not only essential for building blood vessels in the embryo, but also in adulthood, where abnormal VSMC differentiation is implicated in atherosclerosis, hypertension, and restenosis after angioplasty. Interestingly, VSMCs derive from 8 different progenitor cell types and exhibit differing characteristics depending on their origins. Xie and colleagues studied the neural crest cell (NCC) derived VSMCs, which ultimately locate to the aortic arch arteries, ascending aorta and carotid arteries. In vitro studies indicate that growth factor TGFβ regulates differentiation of NCC to VSMC, but in vivo experiments have given contrasting results. Xie and colleagues thus turned to a downstream target of TGFβ—Smad2—to resolve the issue. They deleted Smad2 from NCCs in mice and found that while NCCs still migrated to the aortic arch, they failed to differentiate to VSMCs. The VSMC layer in carotid arteries was also diminished. The team also showed that, in a mouse neural crest cell line, Smad2 interacted with transcription factor MRTFB—an important regulator of NCC to VSMC differentiation—which led to upregulation of VSMC markers. The authors therefore suggest that TGFβ signaling, via Smad2, is indeed important for NCC to VSMC differentiation.Myocardial Efferocytosis Heals the Heart (p 1004)Wan et al identify a macrophage receptor responsible for the disposal of dead heart cells after myocardial infarction.Download figureDownload PowerPointAfter a heart attack, macrophages gather at the injury site to gobble-up dying cardiomyocytes. This feasting on cellular corpses, or efferocytosis, is essential for efficient resolution of inflammation, which in turn limits the degree of adverse cardiac remodeling and the risk of progression to heart failure. Indeed, it has been shown that defective cardiomyocyte clearance in older mice is associated with impaired heart repair, and that macrophages from older animals exhibit reduced efferocytosis. To learn more, Wan and colleagues examined macrophages with deletions of known efferocytosis receptors. They found only one—MERTK—that was essential for removal of necrotic cardiomyocytes through phagocytosis. They also found that mice lacking MERTK still recruited macrophages to their hearts following myocardial infarction. However, inflammation was slow to resolve, there were more dying and dead cardiomyocytes, infarct size was larger, and heart function was worse. Interestingly, while levels of the MERTK receptor increased in the hearts of wild-type mice 3 to 7 days after a myocardial infarction, the authors also found that some of the MERTK was cleaved into an inactive form. Why this inactivation occurs, whether it worsens with age, and whether it can be prevented to improve recovery after myocardial infarction are all avenues the authors plan to investigate.CEACAM1 in Ischemic Stroke (p 1013)Ludewig et al identify an inflammation inhibitor in mice that, after a stroke, limits the breakdown of the blood brain barrier and thus further injury.Download figureDownload PowerPointAfter the initial hypoxic insult of an ischemic stroke, the problems are far from over. The resulting tissue damage leads to recruitment of immune cells, which cause inflammation with possible deterioration of the blood brain barrier and further brain damage. Immune cells express adhesion molecules that allow the cells to stick at the injury site and infiltrate the tissue. Blocking certain adhesion proteins has been shown to reduce cerebral damage after stroke in mice. But Ludewig and colleagues hypothesized that blocking the adhesion molecule CEACAM1 might have the opposite effect—because although it is also expressed on immune cells, it suppresses inflammation. They were right: Mice that lacked CEACAM1 faired worse after experimentally induced stroke. Their infarct sizes were larger, they had more pronounced cerebral inflammation, and their motility and neurological performance were more impaired. It turned out that without the anti-inflammatory action of CEACAM1, more immune cells that produced MMP-9—a secreted protease that promoted the breakdown of the blood brain barrier—were recruited to the ischemic brain. Together the results suggest that boosting CEACAM1 or inhibiting MMP-9 might be attractive ways to limit brain damage after stroke. Previous Back to top Next FiguresReferencesRelatedDetails September 27, 2013Vol 113, Issue 8 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/01.res.0000435046.80748.fd Originally publishedSeptember 27, 2013 PDF download Advertisement

Forgetting to Switch Off SMAD2 in Aneurysmal Disease

Vascular smooth muscle cells (VSMCs) are key component cells of the vascular wall that are critical for contractility of the aorta.1 They respond to biochemical and mechanical signals and play an important role in the regulation of blood pressure. Dysregulation of VSMCs is a hallmark of cardiovascular diseases such as restenosis, hypertension, aneurysms, and atherosclerosis. Investigating the signaling pathways in both normal and dysregulated VSMCs is important for understanding how these cardiovascular diseases develop and for the development of more effective therapies for these potentially life-threatening disorders. Article, see p 881 Aneurysms are dangerous vascular diseases characterized by aortic dilatation with thinning of the medial VSMC layer. Several mechanisms have been implicated in the development of human aortic aneurysms, including the role of transforming growth factor-β1 (TGF-β1), although there are some controversies in the field.2–6 Evidence shows that VSMCs from thoracic aortic aneurysms (TAA) exhibit enhanced activity of SMAD2, a transcription factor that is part of the canonical TGF-β1 pathway and a key effector of the actions of TGF-β1.7 TGF-β1 signaling results in the phosphorylation and translocation of cytoplasmic SMAD2 into the nucleus and consequent transcriptional activation of its target genes.8,9 However, the activation of SMAD2 in TAA has previously been shown to be dissociated from the TGF-β1 pathway. Instead, the SMAD2 hyperactivity is suggested to be attributed to the constitutive overexpression of SMAD2 at the transcriptional level.10 Furthermore, SMAD2 overexpression was associated with key chromatin histone modifications at the SMAD2 promoter, suggesting an element of epigenetic control.10 In the nucleus of mammalian cells, chromosomal DNA is tightly packaged into chromatin, a higher-order structure comprised of subunits called nucleosomes. Each nucleosome consists of an …

Comparison of vascular smooth muscle cells in canine great vessels

BackgroundElucidating the histological characteristics of normal vascular smooth muscle cells (VSMCs) is important for understanding mechanisms of development, disease etiology and the remodeling and/or regeneration process of the vessel. However, knowledge regarding VSMCs is focused primarily on the artery. Although the characteristics of each great vessel are documented, few studies have examined VSMCs in parallel within each great vessel. The present study focused on comparing characteristics of canine VSMCs within the aorta (Ao), branch pulmonary artery (bPA), main pulmonary artery (mPA) and inferior vena cava (IVC), simultaneously.ResultsWestern blot and immunohistochemistry were used to determine VSMC protein content for alpha smooth muscle actin (ASMA), calponin, myosin heavy chain (MHC) and its isozyme SM2, and non-muscle myosin heavy chain B (SMemb). Thickness and ratio of the VSMC layer were also measured. Expression levels of ASMA, calponin and SM2 significantly differed between vessels, except between mPA and either bPA, Ao and IVC vessels. Expression levels of MHC were significantly different in all vessels, whilst expression of SMemb was significantly different in the Ao compared with either bPA and mPA vessels. All vessels were significantly different with respect to total wall and VSMC layer thickness. The ratio between VSMC layer and total wall thickness was significantly different for each vessel, except between bPA and mPA vessels. Histological analysis of the IVC revealed that the VSMC layer does not line evenly and continuously through the long axis or transverse sections. With respect to the pulmonary artery, calponin was expressed to a greater extent in the mPA compared with the bPA (P < 0.01*). In contrast, MHC and SM2 were expressed to a greater extent in the bPA compared with the mPA (P < 0.01*). Differences in VSMC distribution indicate structural differences in the proximal and distal pulmonary artery bifurcation.ConclusionOur results show that the VSMC expression pattern in each great vessel is unique and suggestive of the developmental differences between great vessels. We believe this study provides basic data for the pathology, etiology and regenerative capability of the vessels.