Vessel Smooth Muscle Cells Research Articles

Endothelial-derived nitric oxide impacts vascular smooth muscle cell phenotypes under high wall shear stress condition

The Phenotypic states of vascular smooth muscle cells (SMCs) are essential to understanding vascular pathophysiology. SMCs in vessels generally express a specific set of contractile proteins, but decreased contractile protein expression, indicating a phenotypic shift, is a hallmark of vascular diseases. Recent studies have suggested the relation of abnormally high wall shear stress (WSS) of approximately 20Pa with the aortic disease pathogenesis. However, due to the lack of appropriate experimental models to assess SMC phenotypic states, the details of the phenotypic shift under high WSS conditions remain unclear. In this study, we developed a coculture model where vascular endothelial cells (ECs) were cocultured with SMCs expressing calponin 1, a contractile protein involved in the phenotypic shift of SMCs. We investigated the effects of a pathologically high WSS condition on the phenotypic states of SMCs. Increased calponin 1 expression was found upon exposure to 20Pa WSS compared with a physiological 2Pa condition, whereas the expression of another contractile protein, α-smooth muscle actin (αSMA) remained unchanged. Furthermore, the inhibition of EC-derived nitric oxide (NO), which is associated with endothelial dysfunction in vascular diseases, resulted in a trend of decreasing αSMA and Calponin 1 expression under 20Pa WSS conditions compared with 2Pa. Our findings suggest that EC-derived NO under pathologically high WSS conditions may impact the expression of contractile proteins implicated in aortic pathophysiology.

β-sitosterol alleviates pulmonary arterial hypertension by altering smooth muscle cell phenotype and DNA damage/cGAS/STING signaling

Pulmonary arterial smooth muscle cells (PASMCs) have a neoplastic phenotype characterized by hyperproliferative and anti-apoptotic features that contribute to pulmonary hypertension (PH) development. DNA-sensing adapter protein stimulator of interferon genes (STING) regulate the phenotypic switch of vessel smooth muscle cells. β-sitosterol (SITO) is a nutrient derived from plants that inhibits vascular smooth muscle cell proliferation without notable toxicity. However, the effect of SITO on cancer-like PH-associated pulmonary vascular remodeling and the specific mechanism has not yet be studied. This study investigated the in vitro and in vivo effects of SITO against PH, and its underlying mechanisms. The therapeutic efficacy of SITO was assessed, and its underlying mechanisms were explored in hypoxia-induced and platelet-derived growth factor (PDGF)-BB-stimulated primary PASMCs and in a monocrotaline (MCT)-induced preclinical PH rat model. SITO or sildenafil (SID) were administered after the MCT intraperitoneal injection. Pulmonary parameters, right heart function, morphology, and PASMCs were cultured for verification. The expression levels of DNA damage/cyclic GMP-AMP synthase (cGAS)/STING were determined using immunofluorescence and Western blotting. STING agonists that interfere with PASMCs were used to determine whether STING mediates the effects of SITO. SITO prevented PASMCs proliferation, promoted apoptosis and suppressed phenotypic switching in a dose-dependent manner in vitro and in vivo. In vivo results in rats demonstrated that four weeks of intragastric SITO administration effectively mitigated the MCT-induced elevation of hemodynamic parameters, improved right cardiac function, and reduced pulmonary arteries remodeling. Mechanistically, DNA damage and cGAS/STING/nuclear factor kappa-B signaling activation were observed in rats with PH and cultured PASMCs. SITO exhibited protective effects by suppressing the DNA damage, potentially via inhibiting the expression level of the cGAS/STING signaling pathway. Pharmacological overexpression of STING abolished the anti-proliferative effects of SITO treatment in hypoxia-induced and PDGF-stimulated PASMCs by downregulating PCNA. SITO may be an attractive agent for PH vascular remodeling by inhibiting proliferation and modulating the phenotypic switch in PASMCs via the DNA damage/cGAS/STING signaling pathway. This study provides a novel therapeutic agent and mediator of the pathological development of PASMCs and PH.

A case of giant cell osteosarcoma in a Scottish fold cat

This study describes a case of giant cell osteosarcoma (GCO) localised on the right scapulae in a six-year-old neutered male Scottish Fold cat. GCO is a rare tumour in domestic animals. This tumour is also known as giant cell-rich osteosarcoma. Multinucleated giant cells and osteoid structures are observed in GCO. The mass was 4.6 x 4.4 x 4.3 cm in size and very firm. Microscopically, fusiform spindle-shaped cells and multinucleated giant cells were observed. There were 4-8 mitotic figures in three random high-power fields (400x). Osteoid structures and necrosis were detected in several areas. In these areas, atypia was observed in the cells. Immunohistochemically, vimentin expression was observed in neoplastic cells and multinucleated giant cells. No immunoreactivity against actin and cytokeratin was observed in neoplastic cells. Additionally, vimentin expression was detected in the periphery of blood vessels and actin expression in blood vessel smooth muscle cells. As a result of histopathological and immunohistochemical findings, the mass was determined to be GCO.

Aberrant endothelial expression of hnRNPC1/C2 and VDR and reduced maternal vitamin D levels in women with preeclampsia

Vitamin D deficiency is a widespread health problem globally and vitamin D deficiency/ insufficiency in pregnancy is a risk factor for preeclampsia, a hypertensive disorder in human pregnancy. Vitamin D elicits its biological effects through binding to its receptor VDR. In the present study, we determined maternal vascular expression of VDR and hnRNPC1/C2, a native repressor of VDR, in subcutaneous adipose tissue from women with normal pregnancy and preeclampsia. Maternal antenatal and postnatal vitamin D levels were measured. We found that hnRNPC1/C2 expression was markedly increased, while VDR expression was markedly reduced, in maternal vessel endothelium and smooth muscle cells from women with preeclampsia compared to that from normal pregnant controls. Reduced VDR expression was relevant to low maternal antenatal and postnatal vitamin D levels in women with preeclampsia. Using human umbilical vein endothelial cells (HUVECs) as an endothelial model, we further investigated the role of hnRNPC1/C2-mediated VDR expression in endothelial cells, and tested effect of hnRNPC1/C2 inhibition on endothelial response to bioactive vitamin D, 1,25(OH)2D3. Our results showed that inhibition of hnRNPC1/C2 by hnRNPC1/C2 siRNA resulted in not only an increase in endothelial VDR expression, but further improved endothelial response to 1,25(OH)2D3. These findings indicate that aberrant hnRNPC1/C2 expression may contribute to reduced vascular expression of VDR in women with preeclampsia and suggest that hnRNPC1/C2 could be a target for improving vascular endothelial cell response to vitamin D.

A novel anti-inflammatory role links the CARS2 locus to protection from coronary artery disease

Genome-wide association studies (GWAS) identified a coronary artery disease (CAD) risk locus on 13.q34 tagged by rs61969072 (T/G). This variant lies in an intergenic region, proximal to ING1, CARKD and CARS2 but its causal relationship to CAD is unknown. We first demonstrated that rs61969072 and tightly linked single nucleotide polymorphisms (SNPs) associate with CARS2 but not ING1 or CARKD expression in carotid endarterectomy samples, with reduced CARS2 abundance in carriers of the CAD risk allele (G). THP-1 monocytes were differentiated and polarized to proinflammatory (M1) and anti-inflammatory (M2) macrophages. CARS2 gene expression decreased in M1 and increased in M2 macrophages, consistent with a role for CARS2 in inflammation. Gene expression profiling revealed an increase in pro-inflammatory markers in response to CARS2 siRNA knockdown in THP-1 derived macrophages, accompanied by an increased abundance of inflammatory cytokines in the cell supernatant. Functional enrichment analysis of impacted transcripts identified the anti-inflammatory IL10 signalling pathway. Western blot analysis of CARS2 silenced macrophages revealed reduced STAT3 phosphorylation in response to IL-10 and increased expression of LPS-induced genes that are repressed by IL-10, indicating a role for CARS2 in anti-inflammatory signalling. Finally, to simulate vessel wall conditions, macrophages, and smooth muscle cells (SMC) were maintained in co-culture. Significantly, CARS2 silencing in macrophages altered the SMC phenotype, decreasing expression of contractile genes and increasing expression of inflammatory genes. These data highlight a novel anti-inflammatory novel role for CARS2 in human macrophages and SMCs that may underlie the protective effect of a common GWAS-identified variant.

Immunofluorescence Analysis of NF-kB and iNOS Expression in Different Cell Populations during Early and Advanced Knee Osteoarthritis.

Synovitis of the knee synovium is proven to be a precursor of knee osteoarthritis (OA), leading to a radiologically advanced stage of the disease. This study was conducted to elucidate the expression pattern of different inflammatory factors—NF-kB, iNOS, and MMP-9 in a subpopulation of synovial cells. Thirty synovial membrane intra-operative biopsies of patients (ten controls, ten with early OA, and ten with advanced OA, according to the Kellgren–Lawrence radiological score) were immunohistochemically stained for NF-kB, iNOS, and MMP9, and for different cell markers for macrophages, fibroblasts, leukocytes, lymphocytes, blood vessel endothelial cells, and blood vessel smooth muscle cells. The total number of CD68+/NF-kB+ cells/mm2 in the intima of early OA patients (median = 2359) was significantly higher compared to the total number of vimentin+/Nf-kB+ cells/mm2 (median = 1321) and LCA+/NF-kB+ cells/mm2 (median = 64) (p < 0.001 and p < 0.0001, respectively). The total number of LCA+/NF-kB+ cells/mm2 in the subintima of advanced OA patients (median = 2123) was significantly higher compared to the total number of vimentin+/NF-kB+ cells/mm2 (median = 14) and CD68+/NF-kB+ cells/mm2 (median = 29) (p < 0.0001). The total number of CD68+/iNOS+ cells/mm2 in the intima of both early and advanced OA patients was significantly higher compared to the total number of vimentin+/iNOS+ cells/mm2 and LCA+/iNOS+ cells/mm2 (p < 0.0001 and p < 0.001, respectively). The total number of CD68+/MMP-9+ cells/mm2 in the intima of both early and advanced OA patients was significantly higher compared to the total number of vimentin+/MMP-9+ cells/mm2 and CD5+/MMP-9+ cells/mm2 (p < 0.0001). Macrophages may have a leading role in OA progression through the NF-kB production of inflammatory factors (iNOS and MMP-9) in the intima, except in advanced OA, where leukocytes could have a dominant role through NF-kB production in subintima. The blocking of macrophageal and leukocyte NF-kB expression is a possible therapeutic target as a disease modifying drug.

A Mini Review on Hemostasis and Coagulation

Hemostasis, the cycle by which draining is captured, includes a mind boggling arrangement of physiological and biochemical occasions that end in the development of a steady attachment that seals the vein. The cycle includes connection between the vein divider and platelets, blood coagulation, and fibrinolysis. The endothelium assumes a significant function in obsessive cycles, for example, atherosclerosis, apoplexy, and scattered intravascular coagulation, flawed hemostasis, irritation, invulnerable issues, vascular neoplasia, and metastasis. Following injury, there is expanded entanglement of plasma lipoprotein parts followed by multiplication of blood vessel smooth muscle cells and additionally testimony of free cholesterol and cholesterol esters, anew union of connective tissue, and intracellular and extracellular affidavit of lipids in factor sums. Then again, coagulation continues by an inborn or intravascular pathway and by an outward or tissue juice pathway, the two of which convert prothrombin to thrombin. The vast majority of the properties and elements of Hageman factor apply to plasma thromboplastin predecessor, which additionally assumes a significant part in the underlying initiation phases of coagulation.

Chronic hypoxia in utero increases PAF receptor expression in lung vessels of fetal lambs

The fetus is chronically in low oxygen environment that maintains a desirable high pulmonary vascular resistance (PVR) such that only 8–10% of total cardiac output passes through fetal lungs. With oxygenation, PVR falls dramatically, lung blood flow increases to accommodate the total cardiac output. Chronic hypoxia in utero leads to increase in vessel wall thickness and media wall area in pulmonary arteries of fetal lambs. Platelet activating factor (PAF) is an endogenous lipid molecule with different physiological and pathological properties. In ovine fetal pulmonary VSMC, hypoxia increases PAF receptor (PAFR) density and PAFR binding, indicating that high PAF level and its activities in fetal lungs are, in part, responsible for maintaining the desirable high fetal PVR in utero, but which postnatally manifest as persistent pulmonary hypertension of the newborn (PPHN). In spite of the tremendous advances in therapy of newborn respiratory diseases, PPHN still accounts for about 10–20% of neonatal mortalities. The magnitude and tissue distribution of PAFR expression in pulmonary vasculature of fetal lambs, in vivo, has not been examined. We hypothesized that chronic hypoxia in utero leads to over‐expression of PAFR and PAFR binding in fetal pulmonary vasculature that maintain high fetal PVR in utero. We obtained two groups of term pregnant ewes. One group was raised under low oxygen saturation (high altitude hypoxia, HAH). The other group was raised at sea level (normoxia). Fetuses were exteriorized from anesthetized ewes. Fetal lungs were harvested and sectioned for immunohistochemical staining and analytical microscopy of PAFR protein expression. Lung tissues and pulmonary vessel smooth muscle cells (SMC) were prepared and assayed for PAFR binding. We found that PAFR was expressed in the pulmonary vascular tissues of both groups of fetal lambs. Comparatively, PAFR protein fluorescence was greater in the HAH lung tissues in the internal elastin, endothelium, the smooth muscle layer and the adventitia. Baseline PAFR binding to vessel SMC of the HAH lambs was also greater. These data show that the greater PAFR expression in fetal hypoxic lung environment is in part, responsible for the high PVR of the fetal pulmonary circulation.

A Note on Adenocarcinoma

Hemostasis, the cycle by which draining is captured, includes a mind boggling arrangement of physiological and biochemical occasions that end in the development of a steady attachment that seals the vein. The cycle includes connection between the vein divider and platelets, blood coagulation, and fibrinolysis. The endothelium assumes a significant function in obsessive cycles, for example, atherosclerosis, apoplexy, and scattered intravascular coagulation, flawed hemostasis, irritation, invulnerable issues, vascular neoplasia, and metastasis. Following injury, there is expanded entanglement of plasma lipoprotein parts followed by multiplication of blood vessel smooth muscle cells and additionally testimony of free cholesterol and cholesterol esters, anew union of connective tissue, and intracellular and extracellular affidavit of lipids in factor sums. Then again, coagulation continues by an inborn or intravascular pathway and by an outward or tissue juice pathway, the two of which convert prothrombin to thrombin [1]. The vast majority of the properties and elements of Hageman factor apply to plasma thromboplastin predecessor, which additionally assumes a significant part in the underlying initiation phases of coagulation.

Cellular Distribution of Canonical and Putative Cannabinoid Receptors in Canine Cervical Dorsal Root Ganglia.

Growing evidence indicates cannabinoid receptors as potential therapeutic targets for chronic pain. Consequently, there is an increasing interest in developing cannabinoid receptor agonists for treating human and veterinary pain. To better understand the actions of a drug, it is of paramount importance to know the cellular distribution of its specific receptor(s). The distribution of canonical and putative cannabinoid receptors in the peripheral and central nervous system of dogs is still in its infancy. In order to help fill this anatomical gap, the present ex vivo study has been designed to identify the cellular sites of cannabinoid and cannabinoid-related receptors in canine spinal ganglia. In particular, the cellular distribution of the cannabinoid receptors type 1 and 2 (CB1 and CB2) and putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), and transient receptor potential vanilloid type 1 (TRPV1) have been immunohistochemically investigated in the C6–C8 cervical ganglia of dogs. About 50% of the neuronal population displayed weak to moderate CB1 receptor and TRPV1 immunoreactivity, while all of them were CB2-positive and nearly 40% also expressed GPR55 immunolabeling. Schwann cells, blood vessel smooth muscle cells, and pericyte-like cells all expressed CB2 receptor immunoreactivity, endothelial cell being also PPARα-positive. All the satellite glial cells (SGCs) displayed bright GPR55 receptor immunoreactivity. In half of the study dogs, SGCs were also PPARα-positive, and limited to older dogs displayed TRPV1 immunoreactivity. The present study may represent a morphological substrate to consider in order to develop therapeutic strategies against chronic pain.

Effect of 27nt-miRNA on the differentiation of mesenchymal stem cells into vascular smooth muscle cells

To investigate the effect of 27nt-miRNA on the differentiation of mesenchymal stem cells into vascular smooth muscle cells. The highly expression plasmids of 27nt-miRNA and anti-27nt-miRNA, and negative control plasmids were constructed, packaged with lentivirus and transfected into human umbilical cord mesenchymal stem cells (hUCMSCs). Collagen IV was added to induce hUCMSCs differentiation into blood vessel smooth muscle cells (VSMCs). The cell viability was measured by MTT assay. The expression of SMA, SM22α at mRNA and protein levels was determined by RT-PCR, immunocytochemical staining and Western blotting. Compared with the negative control group, the viability of the 27nt-miRNA overexpression group was decreased by 20.48% (P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was significantly increased (P<0.05); the viability of Anti-27nt-miRNA group was increased 18.07% (P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was decreased (P<0.05). In summary, 27nt-miRNA promotes mesenchymal stem cells differentiation into vascular smooth muscle cells and inhibits cells viability.

The Effect of Voltage‐Sensitive Chloride Channel 6 on Development of Salt‐Sensitive Hypertension

Hypertension is currently the number one risk factor for global disease burden. Advancing our knowledge on the underlying mechanisms of blood pressure (BP) control is essential to reduce the incidence of hypertension. Recent human genome‐wide association studies have linked a number of rare coding mutations and intronic single nucleotide polymorphisms in the gene, CLCN6, to lower average systolic and diastolic blood pressure as well as reduced hypertension risk. The voltage‐gated chloride channel 6 (CLC6) is encoded by CLCN6, which is one of 9 voltage‐gated chloride channel (CLC) family members. The functions of CLC6 at molecular, cellular, and physiological levels are largely unknown.We have previously introduced a 5 amino acid deletion into Clcn6 in the Dahl Salt Sensitive (SS) rat background (SS‐Clcn6em2Mcwim (hereafter called CLC6 KO)). These rats demonstrated lower mean arterial pressure (MAP) compared to WT SS rats (Flister et al., Genome Res, 2013) when placed on a high salt (HS, 4% NaCl) diet for 10 days, suggesting these animals might serve as a model system to study the role of CLCN6 in modulating blood pressure. To further test the role of CLC6 on blood pressure and renal function, we placed CLC6 KO or WT SS rats on a HS diet for 3 weeks and recorded their blood pressure using telemetry. Electrolytes and kidney injury were also analyzed. CLC6 KO rats had a significantly lower BP maintaining a MAP about 15 mmHg lower than the WT SS rats. There were no significant differences in plasma Na+, K+, Cl−, or Ca2+ levels between WT SS and CLC6 KO rats on NS diets; however, the plasma K+ levels of the CLC6 KO rats on the HS diet were significantly elevated compared to controls. Immunohistochemical analysis revealed that CLC6 is strongly expressed in blood vessel smooth muscle cells. Therefore, we studied vasodilatory response of isolated mesenteric resistance arteries in response to increasing concentrations of acetylcholine. We found that arteries from the CLC6 KO animals had impaired dilation to 10 μM acetylcholine, and relaxed to 25 ± 2.9 % of their maximal diameter relative to controls (60 ± 13.9 %). Lastly, we measured spontaneous intracellular Ca2+ activity in intact ex vivo mesenteric artery smooth muscle at single cell resolution using event detection analysis of two‐photon microscopy videos. Cells were constricted with phenylephrine then induced to relax with acetylcholine, and Ca2+ cycling was significantly reduced in the CLC6 KO (19.0 ± 1.7 sec vs 93.0 ± 11.3 sec) during relaxation, consistent with the vessel dilation studies.These results suggest that CLC6 KO is protective against development of salt sensitive hypertension by altering the intracellular Ca2+ activity in arterial smooth muscle cells, but additional studies are needed to more fully understand this effect.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Radiofrequency Ablation of the Atherosclerotic Plaque: a Proof of Concept Study in an Atherosclerotic Model

Increased plaque vascularization is causatively associated with the progression of unstable atherosclerotic vessel disease. We investigated the safety and efficacy of heat-generating radiofrequency ablation (RFA) in reducing the number of vessels in the plaque and adventitia and its effect on plaque size and composition. To this end, New Zealand White rabbits were fed a cholesterol-enriched diet and subjected to balloon denudation of the infrarenal aorta to induce atherosclerotic plaque formation. After 13 weeks, the proximal or distal half of the infrarenal aorta was exposed to transluminal RFA. The untreated half served as an intra-individual control. Optical coherence tomography (OCT) was performed directly after RFA. We found that RFA on the rabbit atherosclerotic plaque is safe and leads to decreased intraplaque vessel density and smooth muscle cell content but does not affect other components of plaque composition or size.

Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells

Serotonin or 5-hydroxytryptamine (5-HT) has been shown to be essential in lots of physiological and pathological processes. It is well known that 5-HT and 5-HT transporter (5-HTT) play important roles in the pulmonary artery in pulmonary hypertension. However, little is known about the function of 5-HTT in other arteries. In this study we found that the expression of 5-HTT was elevated in injured carotid arteries and over-expression of 5-HTT induced proliferation of smooth muscle cells (SMCs); however, this phenotype could be reversed by knocking-down of 5-HTT or endothelial cells conditional medium (EC-CM). A 5-HTT inhibitor, fluoxetine, treated animals also exhibited reduced restenosis after injury. We identified that miR-195 was packaged in the extracellular vesicles from EC-CM. We further confirmed that extracellular vesicles could transfer miR-195 from ECs to SMCs to inhibit the expression of 5-HTT in SMCs and the proliferation of SMCs. These results provide the first evidence that ECs communicate with SMCs via micro-RNA195 in the regulation of the proliferation of SMCs through 5-HTT, which will contribute to a better understanding of communications between ECs and SMCs via micro-RNA. Our findings suggest a potential target for the control of vessel restenosis.

MiRNA-181a/b Regulates Phenotypes of Vessel Smooth Muscle Cells Through Serum Response Factor.

The phenotypic modulation of vessel smooth muscle cells (VSMCs) plays a crucial role in the physiological and pathological conditions of vasculature in response to local environmental changes. The phenotypic transition of VSMCs is largely modulated by the serum response factor (SRF). miR-181a and miR-181b are members of the well-studied miR-181 family and both have complementary sequence in the 3' untranslated region (UTR) of SRF gene. In this article, evidence insinuates that miR-181a/b was involved in VSMCs differentiation through upregulating synthetic marker genes and downregulating contractile ones, respectively. We also confirmed the roles of the miR-181a/b in promoting SMC proliferation, migration, and synthetic phenotype transformation. In addition, miR-181a/b was indicated as directly targeting at 3' UTR of SRF by dual-luciferase assay and Western blot assay. In a word, miR-181a/b is one of the factors involved in VSMC differentiation toward a synthetic phenotype through targeting at SRF. These findings may provide a potential therapeutic approach that miR-181a/b took part in regulating the vessel disorders.

Drug loaded nanoparticle coating on totally bioresorbable PLLA stents to prevent in-stent restenosis.

Biodegradable polymer poly (dl-lactide) (PDLLA) has been used as drug coating material for drug-eluting stents due to its excellent biocompatibility and sustained drug release ability. However, the uniform thin layer drug eluting coating on a stent not only inhibits the blood vessel's smooth muscle cell overgrowth but also delay the endotheliation process which is often associated with the occurrence of acute thrombosis. Therefore, in this study, we developed a novel coating method using PDLLA nanoparticles (NPs) as a coating to overcome this issue. The average 300 nm sized sirolimus-loaded PDLLA nanoparticles were prepared by a conventional emulsion solvent evaporation method. A low temperature plasma polymerization technology to graft hydrophilic polymers on to poly (l-lactide) stent was used to increase the surface coating efficiency of nanoparticles on the stent. Results showed that sirolimus-loaded nanoparticles can be successfully coated on to the stents with sustained drug release properties. In vitro cell culture study showed the drug loaded nanoparticle coating effectively inhibited the proliferation of smooth muscle cells while still allowed a faster proliferation of endothelial cells, suggesting that the new NP coated bioresorbable stents have the potential to reduce both the occurrence of in-stent restenosis and acute thrombosis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 88-95, 2018.

Abstract 10: The Matrix Crosslinking Enzyme Lysyl Oxidase Like-2 as a Target to Reverse Vascular Stiffness

Introduction: Stiffening of the central vasculature is a strong and independent predictor of adverse cardiovascular events. Vascular stiffening is a complex process that involves changes in the vessel wall composition and smooth muscle cell (SMC) function. We recently used an unbiased proteomic approach to identify Lysyl oxidase like 2 (LOXL2) as a potential new target in vascular stiffness. The goal of this study is to characterize the role of LOXL2 in vascular stiffening and its potential as a target to reverse vascular stiffness associated with hypertension. Results: We demonstrate that decreased nitric oxide (NO) bioavailability results in increased secretion and activity of LOXL2 in SMCs. LOXL2 knockdown markedly attenuates SMC adhesion, motility, and proliferation and results in diminished matrix deposition. LOXL2 knockdown also results in striking changes in the stiffness and cytoskeletal remodeling events in CMSs. Tensile testing shows that intact aortas of LOXL2+/- animals are stiffer when compared with those from wild type mice, while there is no difference in decellularized vessels. We next investigated the role of LOXL2 in the development of hypertension using angiotensin II (AngII) infusion in LOXL2+/- (group 1) and wild type (WT; group 2) mice. BP and pulse wave velocity (PWV) increased significantly with AngII infusion in both groups during the study period, without a significant change in heart rate. Compared to WT animals, contractile responsiveness was markedly diminished in LOXL2+/- animals at baseline as well as with AngII infusion when compared with untreated controls. The NO- dependent vasodilatory response to acetylcholine was identical at baseline and diminished significantly with AngII infusion in both groups of animals. There was no difference between the groups in the endothelium-independent response to sodium nitroprusside. Conclusion: In this study, we demonstrated the role of NO in the regulation of LOXL2. Interestingly, LOXL2 appears to have a dual role in vascular stiffness by affecting both SMC function as well as matrix composition. We therefore conclude that LOXL2 is a novel target involved in vascular stiffness that requires further characterization to elicit the possibility of therapeutic intervention.

Variants in ALOX5, ALOX5AP and LTA4H are not associated with atherosclerotic plaque phenotypes: The Athero-Express Genomics Study

The eicosanoid genes ALOX5, ALOX5AP and LTA4H have been implicated in atherosclerosis. We assessed the impact of common variants in these genes on gene expression, circulating protein levels, and atherosclerotic plaque phenotypes. We included patients from the Stockholm Atherosclerosis Gene Expression study (STAGE, N = 109), and the Athero-Express Biobank Study (AE, N = 1443). We tested 1453 single-nucleotide variants (SNVs) in ALOX5, ALOX5AP and LTA4H for association with gene expression in STAGE. We also tested these SNVs for association with seven histologically defined plaque phenotypes in the AE (which included calcification, collagen, cellular content, atheroma size, and intraplaque vessel density and hemorrhage). We replicate a known cis-eQTL (rs6538697, p = 1.96 × 10(-6)) for LTA4H expression in whole blood of patients from STAGE. We found no significant association for any of the SNVs tested with serum levels of ALOX5 or ALOX5AP (p > 5.79 × 10(-4)). For atherosclerotic plaque phenotypes the strongest associations were found for intraplaque vessel density and smooth muscle cells in the ALOX5AP locus (p > 1.67 × 10(-4)). We replicate a known eQTL for LTA4H expression in whole blood using STAGE data. We found no associations of variants in and around ALOX5, ALOX5AP and LTA4H with serum ALOX5 or ALOX5AP levels, or plaque phenotypes. On the supposition that these genes play a causal role in atherosclerosis, these results suggest that common variants in these loci play a limited role (if any) in influencing advanced atherosclerotic plaque morphology to the extent that it impacts atherosclerotic disease.

Immunohistochemical evaluation of androgen receptor and nerve structure density in human prepuce from patients with persistent sexual side effects after finasteride use for androgenetic alopecia.

Finasteride is an inhibitor of 5-α-reductase used against male androgenetic alopecia (AGA). Reported side effects of finasteride comprise sexual dysfunction including erectile dysfunction, male infertility, and loss of libido. Recently these effects were described as persistent in some subjects. Molecular events inducing persistent adverse sexual symptoms are unexplored. This study was designed as a retrospective case-control study to assess if androgen receptor (AR) and nerve density in foreskin prepuce specimens were associated with persistent sexual side effects including loss of sensitivity in the genital area due to former finasteride use against AGA. Cases were 8 males (aged 29–43 years) reporting sexual side effects including loss of penis sensitivity over 6 months after discontinuation of finasteride who were interviewed and clinically visited. After informed consent they were invited to undergo a small excision of skin from prepuce. Controls were 11 otherwise healthy matched men (aged 23–49 years) who undergone circumcision for phimosis, and who never took finasteride or analogues. Differences in AR expression and nerve density in different portions of dermal prepuce were evaluated in the 2 groups. Density of nuclear AR in stromal and epithelial cells was higher in cases (mean 40.0%, and 80.6% of positive cells, respectively) than controls (mean 23.4%, and 65.0% of positive cells, respectively), P = 0.023 and P = 0.043, respectively. Conversely, percentage of vessel smooth muscle cells positive for AR and density of nerves were similar in the 2 groups. The ratio of AR positive stromal cells % to serum testosterone concentrations was 2-fold higher in cases than in controls (P = 0.001). Our findings revealed that modulation of local AR levels might be implicated in long-term side effects of finasteride use. This provides the first evidence of a molecular objective difference between patients with long-term adverse sexual effects after finasteride use versus drug untreated healthy controls in certain tissues.

Interacting influence of diuretics and diet on BK channel-regulated K homeostasis

Large conductance, Ca-activated K channels (BK) are abundantly located in cells of vasculature, glomerulus, and distal nephron, where they are involved in maintaining blood volume, blood pressure, and K homeostasis. In mesangial cells and smooth muscle cells of vessels, the BK-α pore associates with BK-β1 subunits and regulates contraction in a Ca-mediated feedback manner. The BK-β1 also resides in connecting tubule cells of the nephron. BK-β1 knockout mice (β1KO) exhibit fluid retention, hypertension, and compromised K handling. The BK-α/β4 resides in acid/base transporting intercalated cells (IC) of the distal nephron, where they mediate K secretion in mammals on a high K, alkaline diet. BK-α expression in IC is increased by a high K diet via aldosterone. The BK-β4 subunit and alkaline urine are necessary for the luminal expression and function of BK-α in mouse IC. In distal nephron cells, membrane BK-α expression is inhibited by WNK4 in in vitro expression systems, indicating a role in the hyperkalemic phenotype in patients with familial hyperkalemic hypertension type 2 (FHHt2). β1KO and BK-β4 knockout mice (β4KO) are hypertensive because of exaggerated epithelial Na channels (ENaC) mediated Na retention in an effort to secrete K via only renal outer medullary K channels (ROMK). BK hypertension is resistant to thiazides and furosemide, and would be more amenable to ENaC and aldosterone inhibiting drugs. Activators of BK-α/β1 or BK-α/β4 might be effective blood pressure lowering agents for a subset of hypertensive patients. Inhibitors of renal BK would effectively spare K in patients with Bartter Syndrome, a renal K wasting disease.