Rat Smooth Muscle Cells Research Articles

Abstract 20966: Increasing Cav1.2 in Smooth Muscle Cells Has Different Effects on Vascular Remodeling Dependent of Insults

Introduction: Increasing Calcium (Ca 2+ ) influx through the L-type Ca 2+ channel (Cav1.2) increases smooth muscle cell (SMC) contractility while the loss of Cav1.2 is associated with enhanced SMC remodeling. However, increasing Ca 2+ influx into SMCs through non-Cav1.2 routes is often associated with enhanced vascular remodeling under pathological conditions. Whether increasing Cav1.2 in SMCs may inhibit or promote SMC remodeling is unclear. Hypothesis: Increasing Cav1.2 in SMC protects SMCs from remodeling. Methods: Transgenic mice with SMC-specific overexpression of Cavβ2a and control mice were subjected to carotid artery ligation or aneurysm induction (high fat feeding for 2 months and then HFD+AngII infusion for 1 month) to study vascular remodeling. Results: 1. The Cav1.2 current in SMCs from middle cerebral artery was increased by 98.5±5.4%; 2. The left common carotid artery (LCCA) close to the internal and external arterial bifurcation were completely ligated. After ligation for 3 weeks, there were less thickening of the media and less neointima formation in LCCA region proximal to the ligation in TG (n=8) than in control (n=8) mice; in accordance, there were less EdU labelled cells in TG. The surface area of TG SMCs was not increased as such as in the control. 3. HFD+AngII induces higher frequency (10/10 in TG vs. 4/13 in control) of aneurysm/aortic dilation segments. Among the mice with aortic aneurysm/dilation, the average number of segments of aneurysm/arotic dilation in each mouse was higher in TG (2.7±0.2) than in control (1.3±0.2) mice. The TG mice had thinner vascular wall, larger aneurysmal area accompanied with more severe elastin and middle layer degradation, and more inflammatory cell infiltration in the aortic aneurysm/dilation region than control mice. 4. Rat smooth muscle cells in culture infected with Cavβ2a-GFP adenoviruses (MOI=10) had less proliferation (both total number of cells and EdU + cells) but more death (LDH and TUNEL+) than rat SMCs infected with AdGFP (MOI=10). Less collagen synthesis was observed in rat SMCs infected with Ad Cavβ2a-GFP. Conclusions: Increased Cav1.2 in SMCs may inhibit or promote vascular smooth muscle remodeling depending on the injury nature.

Regulation of Coronary Arterial Large Conductance Ca2+-Activated K+ Channel Protein Expression and Function by n-3 Polyunsaturated Fatty Acids in Diabetic Rats

Aim: The objective of this study was to examine the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) on coronary arterial large conductance Ca²⁺-activated K⁺ (BK) channel function in coronary smooth muscle cells (SMCs) of streptozotocin-induced diabetic rats. Methods: The effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on coronary BK channel open probabilities were determined using the patch clamp technique. The mRNA and protein expressions of BK channel subunits were measured using qRT-PCR and Western blots. The coronary artery tension and coronary SMC Ca²⁺ concentrations were measured using a myograph system and fluorescence Ca²⁺ indicator. Results: Compared to nondiabetic control rats, the BK channel function was impaired with a reduced response to EPA and DHA in freshly isolated SMCs of diabetic rats. Oral administration of n-3 PUFAs had no effects on protein expressions of BK channel subunits in nondiabetic rats, but significantly enhanced those of BK-β₁ in diabetic rats without altering BK-α protein levels. Moreover, coronary ring tension induced by iberiotoxin (a specific BK channel blocker) was increased and cytosolic Ca²⁺ concentrations in coronary SMCs were decreased in diabetic rats, but no changes were found in nondiabetic rats. Conclusions: n-3 PUFAs protect the coronary BK channel function and coronary vasoreactivity in diabetic rats as a result of not only increasing BK-β₁ protein expressions, but also decreasing coronary artery tension and coronary smooth muscle cytosolic Ca²⁺ concentrations.

Activation of aldehyde dehydrogenase 2 slows down the progression of atherosclerosis via attenuation of ER stress and apoptosis in smooth muscle cells.

Aldehyde dehydrogenase 2 (ALDH2) is a key mitochondrial enzyme in the metabolism of aldehydes and may have beneficial cardiovascular effects for conditions such as cardiac hypertrophy, heart failure, myocardial I/R injury, reperfusion, arrhythmia, coronary heart disease and atherosclerosis. In this study we investigated the role of ALDH2 in the progression of atherosclerosis and the underlying mechanisms, with a focus on endoplasmic reticulum (ER) stress. A clinical study was performed in 248 patients with coronary heart disease. The patients were divided into two groups according to their ALDH2 genotype. Baseline clinical characteristics and coronary angiography were recorded, and the coronary artery Gensini score was calculated. Serum levels of 4-hydroxy-2-nonenal (4-HNE) were detected. The clinical study revealed that the mutant ALDH2 genotype was an independent risk factor for coronary heart disease. ALDH2 gene polymorphism is closely associated with atherosclerosis and the severity of coronary artery stenosis. Serum levels of 4-HNE were significantly higher in patients with the mutant ALDH2 genotype than in patients with the wild-type ALDH2 genotype. As an in vitro model of atherosclerosis, rat smooth muscle cells (SMCs) were treated with oxygenized low-density lipoprotein (ox-LDL), which significantly elevated the levels of ER markers glucose-regulated protein78 (GRP78), protein kinase R-like ER kinase (PERK), phosphorylated eukaryotic translation initiation factor α subunit (p-eIF2α), activating transcription factor-4 (ATF-4), CEBP homologous protein (CHOP) and 4-HNE in the cells. All the ox-LDL-induced responses were significantly attenuated in the presence of Alda-1 (an ALDH2 activating agent), and accentuated in the presence of daidzin (an ALDH2 inhibitor). Furthermore, pretreatment with ALDH2 activator Alda-1 significantly decreased ox-LDL-induced apoptosis. Similarly, overexpression of ALDH2 protected SMCs against ox-LDL-induced ER stress as well as ER stress-induced apoptosis. These findings suggest that ALDH2 may slow the progression of atherosclerosis via the attenuation of ER stress and apoptosis in smooth muscle cells.

Oxidative stress enhances and modulates protein S-nitrosation in smooth muscle cells exposed to S-nitrosoglutathione

Among S-nitrosothiols showing reversible binding between NO and -SH group, S-nitrosoglutathione (GSNO) represents potential therapeutics to treat cardiovascular diseases (CVD) associated with reduced nitric oxide (NO) availability. It also induces S-nitrosation of proteins, responsible for the main endogenous storage form of NO. Although oxidative stress parallels CVD development, little is known on the ability of GSNO to restore NO supply and storage in vascular tissues under oxidative stress conditions.Aortic rat smooth muscle cells (SMC) were stressed in vitro with a free radical generator (2,2′-azobis(2-amidinopropane) dihydrochloride, AAPH). The cellular thiol redox status was reflected through levels of reduced glutathione and protein sulfhydryl (SH) groups. The ability of GSNO to deliver NO to SMC and to induce protein S-nitrosation (investigated via mass spectrometry, MS), as well as the implication of two redox enzymes involved in GSNO metabolism (activity of gamma-glutamyltransferase, GGT, and expression of protein disulfide isomerase, PDI) were evaluated.Oxidative stress decreased both intracellular glutathione and protein -SH groups (53% and 32% respectively) and caused a 3.5-fold decrease of GGT activity, while PDI expression at the plasma membrane was 1.7-fold increased without any effect on extracellular GSNO catabolism. Addition of GSNO (50 μM) increased protein -SH groups and protein S-nitrosation (50%). Mass spectrometry analysis revealed a higher number of S-nitrosated proteins under oxidative stress (83 proteins, vs 68 in basal conditions) including a higher number of cytoskeletal proteins (15, vs 9 in basal conditions) related with cell contraction, morphogenesis and movement. Furthermore, proteins belonging to additional protein classes (cell adhesion, transfer/carrier, and transporter proteins) were S-nitrosated under oxidative stress.In conclusion, higher levels of GSNO-dependent S-nitrosation of proteins from the cytoskeleton and the contractile machinery were identified under oxidative stress conditions. The findings may prompt the identification of suitable biomarkers for the appraisal of GSNO bioactivity in the CVD treatment.

Effect and mechanism of intermittent alkaline stimulation on high phosphorus induced calcification in vascular smooth muscle cells of rats

Objective: To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus. Methods: VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus+ pH7.4, high phosphorus+ pH7.5, high phosphorus+ pH7.6 and high phosphorus+ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L β-glycerophosphate and alkalized by 7.4% NaHCO(3) to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel β(3) subunit(LTCC β(3)) and Runt related transcription factor 2 (Runx2) were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase (ALP) activity was measured by enzyme linked immunosorbent assay (ELISA) and the calcification was observed by measuring calcium content. Results: (1) Compared with control group, the gene and protein expressions of LTCC β(3) were higher in high phosphorus+ pH7.4 group (0.49±0.03 vs. 0.23±0.02 and 0.45±0.03 vs. 0.26±0.02 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.86±0.05) and protein(0.62±0.04) expressions of LTCC β(3) were higher in high phosphorus+ pH7.5 group (P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.99±0.05) and protein(0.80±0.03) expressions of LTCC β(3) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.16±0.05) and protein(0.93±0.03) expressions of LTCC β(3) were higher in high phosphorus+ pH7.7 group (all P<0.05). (2) Compared with control group, calcium ion influx were higher in high phosphorus+ pH7.4 group (124.61±6.06 vs. 75.68±7.82, P<0.05). Compared with high phosphorus+ pH7.4 group, calcium ion influx was higher in high phosphorus+ pH7.5 group(210.85±9.75, P<0.05). Compared with high phosphorus+ pH7.5 group, calcium ion influx was higher in high phosphorus+ pH7.6 group(298.44±11.42, P<0.05). Compared with high phosphorus+ pH7.6 group, calcium ion influx was higher in high phosphorus+ pH7.7 group(401.13±11.41, P<0.05). (3) Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus+ pH7.4 group (0.60±0.04 vs. 0.34±0.03, 0.42±0.04 vs. 0.21±0.02, 67.2±4.3 vs. 23.2±2.3 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.76±0.05) and protein(0.68±0.03) expressions of Runx2 and ALP(102.1±5.4) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.90±0.05) and protein(0.90±0.05) expressions of Runx2 and ALP(139.3±4.9) were higher in high phosphorus+ pH7.6 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.11±0.05) and protein(1.08±0.06) expressions of Runx2 and ALP(197.0±6.7) were higher in high phosphorus+ pH7.7 group (all P<0.05). (4) Compared with control group, the calcium content were higher in high phosphorus+ pH7.4 group ((75.4±4.3)mg/g pro vs.(25.2±2.1)mg/g pro, P<0.05). Compared with high phosphorus+ pH7.4 group, the calcium content were higher in high phosphorus+ pH7.5 group ((100.8±5.7) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.5 group, the calcium content were higher in high phosphorus+ pH7.6 group ((143.5±6.1) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.6 group, the calcium content were higher in high phosphorus+ pH7.7 group ((205.1±8.2) mg/g pro, P<0.05). Conclusion: Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC β(3) subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.

Regional functional heterogeneity of aortic smooth muscle cells in rats

The aorta is a magistral artery, which has been traditionally looked upon as a vessel whose properties are invariable throughout its length. However, in the most recent decade, there have been accumulated data that provide evidence that different aorta sections arise from different embryonic origins and that the population of smooth muscle cells making up the vessel’s wall is, consequently, heterogenic. Tracing the fate of smooth muscle cells, the basic components of the vessel, with the aid of genetic marking methods revealed that the cells’ response to various factors is largely determined by the embryonic origin of a certain cell population. However, functional differences between the smooth muscle cells making up different aorta sections remain poorly understood. The aim of the current work was to compare the functional characteristics of the populations of aortic wall smooth muscle cells obtained from the aorta sections differing by their embryonic origin. Towards this end, we obtained smooth muscle cell cultures from the three aorta sections of linear rats, namely, the neural crest derived ascending thoracic aorta, the somites derived descending thoracic aorta, and splanchnic mesoderm derived abdominal aorta. Using immunocytochemistry and Western blotting, the cells from the different regions of aorta were compared on the basis of smooth muscle actin, vimentin, and SM22 content in them. Cell proliferation rate was estimated using the growth curves method. We have demonstrated that the three smooth muscle cell populations arising from different embryonic origins differ in their morphological characteristics as well as by smooth muscle actin and SM22 content. We have shown that smooth muscle cells from the ascending aorta proliferate more actively than the corresponding cells from the descending thoracic aorta. Thus, the functional properties of the populations of rat aortic smooth muscle cells are different and depend on the embryonic origin of the aorta section from which they were obtained.

Abstract 332: Inhibition of the Enhancer of Zeste Homolog Family Mitigates Intimal Hyperplasia in Rat Carotid Arteries

Rationale: The enhancer of zeste homologs 1 and 2 (EZH1 and EZH2) are histone-lysine N-methyltransferases that modify histones by methylation which leads to transcriptional repression. It has recently been shown that EZH1 and EZH2 play an important role in various cancers. Whether they play a role in recurrent vascular diseases is not known. Objective: We assessed whether EZH1 and EZH2 are important contributors to the development of intimal hyperplasia (IH) and restenosis. Methods and Results: Following rat carotid balloon angioplasty, EZH2 showed highest expression at day 3 post procedure and then decreased at day 7 in injured arteries, as determined by Western blotting. Change of EZH1 is less prominent. Dual inhibition of EZH1 and EZH2 through peri-adventitial administration of a selective inhibitor, UNC-1999, effectively inhibited intimal hyperplasia, with ~40% reduction in the ratio of intima to media (I/M). Moreover, in cultured primary rat smooth muscle cells (SMCs) as well as MOVAS cells, pretreatment with 5μM UNC-1999 resulted in a 60% decrease in cell proliferation and ~80% reduction of migration that were stimulated by PDGF-BB. Simultaneous knockdown of EZH1 and EZH2, as well as knockdown of their shared scaffold protein(EED), led to effective inhibition of proliferation and migration of SMCs. However, knockdown of EZH2 alone did not recapitulate the effects of dual inhibition of EZH1 and EZH2. Conclusions: This study demonstrates that dual inhibition of EZH1 and EZH2, both histone modifiers, mitigates intimal hyperplasia in vivo and attenuates PDGF-BB stimulated SMC proliferation and migration in vitro.

Usefulness of Plasma Tissue Inhibitor of Matrix Metalloproteinase-4 to Predict Death and Myocardial Infarction in Patients With Diabetes Mellitus Referred for Coronary Angiography

TIMP-4 is the newest member of a family of secreted proteins known as the tissue inhibitors of metalloproteases that selectively inhibit matrix metalloproteases. TIMP-4 is abundantly expressed in human cardiovascular structures and has been implicated in cardiovascular disease. Furthermore, it has also been shown to be a novel target of peroxisome proliferator-activated receptor gamma in rat smooth muscle cells, suggesting a potential role in diabetes mellitus as well. However, there have been no studies that have specifically examined the utility of baseline plasma TIMP-4 levels for the prediction of long-term adverse cardiovascular outcomes. In this study, baseline plasma TIMP-4 levels were measured in 162 male patients with diabetes mellitus who were referred for coronary angiography and followed prospectively for the development of all-cause mortality and enzymatically confirmed myocardial infarction (MI) out to 5years. After adjustment for a variety of baseline clinical, angiographic and laboratory parameters, plasma TIMP-4 levels were an independent predictor of all-cause mortality (hazard ratio 1.60, 95% CI 1.13 to 2.26; p= 0.0082) and MI (hazard ratio 1.61, 95% CI 1.19 to 2.18; p= 0.0021) at 5years. Furthermore, in additional multivariate models that adjusted for a variety of biomarkers with established prognostic efficacy, TIMP-4 remained an independent predictor of adverse outcomes. In conclusion, elevated levels of TIMP-4 are associated with an increased risk of long-term all-cause mortality and MI in patients with diabetes mellitus referred for coronary angiography. Moreover, this association is independent of a variety of clinical, angiographic, and laboratory variables, including biomarkers with established prognostic efficacy in the prediction of adverse cardiovascular outcomes.

TASK-1 potassium channel is not critically involved in mediating hypoxic pulmonary vasoconstriction of murine intra-pulmonary arteries

The two-pore domain potassium channel KCNK3 (TASK-1) is expressed in rat and human pulmonary artery smooth muscle cells. There, it is associated with hypoxia-induced signalling, and its dysfunction is linked to pathogenesis of human pulmonary hypertension. We here aimed to determine its role in hypoxic pulmonary vasoconstriction (HPV) in the mouse, and hence the suitability of this model for further mechanistic investigations, using appropriate inhibitors and TASK-1 knockout (KO) mice. RT-PCR revealed expression of TASK-1 mRNA in murine lungs and pre-acinar pulmonary arteries. Protein localization by immunohistochemistry and western blot was unreliable since all antibodies produced labelling also in TASK-1 KO organs/tissues. HPV was investigated by videomorphometric analysis of intra- (inner diameter: 25–40 μm) and pre-acinar pulmonary arteries (inner diameter: 41–60 μm). HPV persisted in TASK-1 KO intra-acinar arteries. Pre-acinar arteries developed initial HPV, but the response faded earlier (after 30 min) in KO vessels. This HPV pattern was grossly mimicked by the TASK-1 inhibitor anandamide in wild-type vessels. Hypoxia-provoked rise in pulmonary arterial pressure (PAP) in isolated ventilated lungs was affected neither by TASK-1 gene deficiency nor by the TASK-1 inhibitor A293. TASK-1 is dispensable for initiating HPV of murine intra-pulmonary arteries, but participates in sustained HPV specifically in pre-acinar arteries. This does not translate into abnormal rise in PAP. While there is compelling evidence that TASK-1 is involved in the pathogenesis of pulmonary arterial hypertension in humans, the mouse does not appear to serve as a suitable model to study the underlying molecular mechanisms.

The Epac protein inhibitor ESI-09 eliminates the tonic phase of aorta contraction induced by endogenic vasoconstrictors in rats

It has been shown that the Epac1 and Epac2 protein inhibitor ESI-09 has no effect on the amplitude of contraction of aortic rings caused by the influence of serotonin, noradrenaline, or KCl depolarizing solution, but changes the kinetics of the contractile response. It was noted that in the presence of ESI-09 the curve of the relaxation phase in intact and deendothelized vessels moved to the left under the impact of serotonin or KCl and the phase of prolonged tonic contraction, which developed after the exposure to noradrenalin, was canceled. It was found that ESI-09 exerted different effects on the induced growth in the concentration of cytoplasmic Ca2+ in the aortic smooth muscle cells of rats depending on the agonist, whereas the selective inhibitor Epac2 ESI-05 has no effect on vascular contractility and calcium metabolism in the aortic smooth muscle of rats. The cAMP-independent participation of Epac1 in the formation of the contractile response to the influence of vasoconstrictor compounds was revealed.

Gene Expressions Underlying Mishandled Calcium Clearance and Elevated Generation of Reactive Oxygen Species in the Coronary Artery Smooth Muscle Cells of Chronic Heart Failure Rats.

Background:The calcium clearance and reactive oxygen species (ROS) generations in the coronary artery smooth muscle cells in chronic heart failure (HF) have not been fully investigated. Therefore, we attempted to understand the gene expressions underlying the mishandling of calcium clearance and the accumulations of ROS.Methods:We initially established an animal model of chronic HF by making the left anterior descending coronary artery ligation (CAL) in rats, and then isolated the coronary artery vascular smooth muscle cells from the ischemic and the nonischemic parts of the coronary artery vessels in 12 weeks after CAL operation. The intracellular calcium concentration and ROS level were measured using flow cytometry, and the gene expressions of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a), encoding sarcoplasmic reticulum Ca2+-ATPase 2a, encoding sodium-calcium exchanger (NCX), and p47phox encoding a subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were examined using real-time quantitative reverse transcription polymerase chain reaction and Western blotting, respectively.Results:We found that the calcium accumulation and ROS generation in the coronary artery smooth muscle cells isolated from either the ischemic or the nonischemic part of the CAL coronary artery vessel were significantly increased irrespective of blood supply (all P < 0.01). Moreover, these were accompanied by the increased expressions of NCX and p47phox, the decreased expression of SERCA2a, and the increased amount of phosphorylated forms of p47phox in NADPH oxidase (all P < 0.05).Conclusions:Our results demonstrated that the disordered calcium clearance and the increased ROS generation occurred in the coronary artery smooth muscle cells in rats with chronic HF produced by ligation of the left anterior descending coronary artery (CAL), and which was found to be disassociated from blood supply, and the increased generation of ROS in the cells was found to make concomitancy to the increased activity of NADPH oxidase in cytoplasm.

Hemoglobin induced cell trauma indirectly influences endothelial TLR9 activity resulting in pulmonary vascular smooth muscle cell activation.

It is now well established that both inherited and acquired forms of hemolytic disease can promote pulmonary vascular disease consequent of free hemoglobin (Hb) induced NO scavenging, elevations in reactive oxygen species and lipid peroxidation. It has recently been reported that oxidative stress can activate NFkB through a toll-like receptor 9 (TLR9) mediated pathway; further, TLR9 can be activated by either nuclear or mitochondrial DNA liberated by stress induced cellular trauma. We hypothesis that Hb induced lipid peroxidation and subsequent endothelial cell trauma is linked to TLR9 activation, resulting in IL-6 mediated pulmonary smooth muscle cell proliferation. We examined the effects of Hb on rat pulmonary artery endothelial and smooth muscle cells (rPAEC and rPASMC, respectively), and then utilized TLR9 and IL6 inhibitors, as well as the Hb and heme binding proteins (haptoglobin (Hp) and hemopexin (Hpx), respectively) to further elucidate the aforementioned mediators. Further, we explored the effects of Hb in vivo utilizing endothelial cell (EC) specific myeloid differentiation primary response gene-88 (MyD88) and TLR9 null mice. Our data show that oxidized Hb induces lipid peroxidation, cellular toxicity (5.5 ± 1.7 fold; p≤0.04), increased TLR9 activation (60%; p = 0.01), and up regulated IL6 expression (1.75±0.3 fold; p = 0.04) in rPAEC. Rat PASMC exhibited a more proliferative state (13 ± 1%; p = 0.01) when co-cultured with Hb activated rPAEC. These effects were attenuated with the sequestration of Hb or heme by Hp and Hpx as well as with TLR9 an IL-6 inhibition. Moreover, in both EC-MyD88 and TLR9 null mice Hb-infusion resulted in less lung IL-6 expression compared to WT cohorts. These results demonstrate that Hb-induced lipid peroxidation can initiate a modest TLR9 mediated inflammatory response, subsequently generating an activated SMC phenotype.

Chai-Qin-Cheng-Qi Decoction and Carbachol Improve Intestinal Motility by Regulating Protein Kinase C-Mediated Ca2+ Release in Colonic Smooth Muscle Cells in Rats with Acute Necrotising Pancreatitis.

Chai-Qin-Cheng-Qi decoction (CQCQD) improves intestinal motility in acute pancreatitis (AP), but the mechanism(s) require elucidation. We investigated the effects of CQCQD and carbachol, a prokinetic agent, on colonic smooth muscle cells (SMCs) in L-arginine-induced necrotising AP model in rats. In treatment groups, intragastric CQCQD (20 g/kg, 2 hourly × 3 doses) or intraperitoneal carbachol (60 μg/kg) was given 24 hours after induction of AP. Both CQCQD and carbachol decreased the severity of pancreatic and colonic histopathology (all P < 0.05). Both CQCQD and carbachol reduced serum intestinal fatty acid binding protein, vasoactive intestinal peptide, and substance P and increased motility levels. CQCQD upregulated SMC phospholipase C-beta 1 (PLC-β1) mRNA and PLC protein (both P < 0.05), while both treatments upregulated protein kinase C-alpha (PKC-α) mRNA and PKC protein and downregulated adenylate cyclase (AC) mRNA and protein compared with no treatment (all P < 0.05). Neither treatment significantly altered L-arginine-induced PKC-β1 and PKC-ε mRNA reduction. Both treatments significantly increased fluorescence intensity of SMC intracellular calcium concentration [Ca2+]i (3563.5 and 3046.9 versus 1086.9, both P < 0.01). These data suggest CQCQD and carbachol improve intestinal motility in AP by increasing [Ca2+]i in colonic SMCs via upregulating PLC, PKC and downregulating AC.

Nrf2 Activation Attenuates Hemolysis but Promotes Pulmonary Vascular Dysfunction in Sickle Mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major transcription factor that coordinates the body’s antioxidant and cytoprotective defense against a variety of toxins. Several Nrf2 activators can reactivate gamma globin gene expression and augment fetal hemoglobin production. More recently, genetic and pharmacologic evidence have shown that Nrf2 activation can specifically mitigate the severity of hemolytic anemia, and systemic and local inflammation in transgenic sickle cell disease (SCD) mice. Based on these encouraging results Nrf2 activation has emerged as an attractive therapeutic strategy in SCD. However, the BEACON trial of the Nrf2 activator CDDO-Methyl Ester (CDDO-Me) showed that this therapeutic approach can cause adverse cardiovascular events in patients with chronic kidney disease with comorbid diabetes. Hitherto, the efficacy-toxicity profile generated by individual Nrf2 activating drugs has not been investigated in SCD. There are hundreds of synthetic and naturally occurring Nrf2 activating compounds, and each class of Nrf2 activating compound has a unique pharmacokinetic, pharmacodynamic, toxicokinetic and toxicodynamic profile. We have recently demonstrated that intravascular hemolysis deteriorates with aging in transgenic sickle (SS) mice in a process that can be mitigated by the Nrf2 activator 3H-1,2-dithiole-3-thione (D3T) (Ghosh et al., JCI Insight, 2016). In this study, an in vitro screen of five Nrf2 activating compounds revealed CDDO-Me to be the most potent inducer of cytoprotective enzymes in human pulmonary microvascular endothelial cells. Thus, we performed a long-term prophylactic CDDO-Me treatment of SS mice and examined the effect of the drug on intravascular hemolysis and vascular dysfunction. A cohort of newly weaned SS mice aged ~4 weeks were randomly assigned to receive CDDO-Me (20µmoles/kg/TIW, n=6) or Vehicle (DMSO/TIW, n=10) by oral gavage for 4 months. After the treatment, the total hemoglobin increased by 10% in the CDDO-Me group while it decreased by 5% in the vehicle-treated group (p<0.05). Plasma concentration of VCAM-1 increased in both groups compared to their respective baseline (vehicle p<0.001; CDDO-Me p<0.05). The lack of impact on VCAM-1 by CDDO-Me was contrary to our results with D3T (Ghosh et al., JCI Insight, 2016). To examine this further, we performed vascular reactivity studies on isolated pulmonary arteries. Second order pulmonary arteries were isolated, cut into 2mm rings, and mounted using 40 μm wire. Rings were set at a resting tension equivalent to 100 mmHg transmural pressure. Cumulative dose response curves of endothelin-1 (100 pM-10 nM) and acetylcholine (10nM-100 μM) were used to study vasoconstriction and vasodilation, respectively. Pulmonary arteries isolated from the CDDO-Me treated SS mice showed a 15% reduction in acetylcholine stimulated vasodilation compared to the arteries isolated from the vehicle treated littermates (vehicle n=5, CDDO-Me n=5; p<0.001). In vitro studies of rat smooth muscle cells revealed significant up-regulation of endothelin receptor subtype A (ETA) mRNA by CDDO-Me (p < 0.01, n=6). We obtained similar results of increased ETA expression in rat smooth muscle cells treated with another Nrf2 activator, Dimethyl Fumarate (DMF) (p < 0.01, n=6). Upregulation of endothelin or the ETA is frequently found in pulmonary hypertension. The hypertensive phenotype that we report here in SS mice treated with CDDO-Me is consistent with the adverse cardiovascular events found in the BEACON trial. Together our results show that while CDDO-Me can stabilize and potentially improve hemolytic anemia in SCD, this drug and potentially other Nrf2 activators may promote the development of pulmonary hypertension, which is a major adult complication of SCD. DisclosuresNo relevant conflicts of interest to declare.

BK channels in rat and human pulmonary smooth muscle cells are BKα-β1 functional complexes lacking the oxygen-sensitive stress axis regulated exon insert.

A loss of K+ efflux in pulmonary arterial smooth muscle cells (PASMCs) contributes to abnormal vasoconstriction and PASMC proliferation during pulmonary hypertension (PH). Activation of high-conductance Ca2+-activated (BK) channels represents a therapeutic strategy to restore K+ efflux to the affected PASMCs. However, the properties of BK channels in PASMCs-including sensitivity to BK channel openers (BKCOs)-are poorly defined. The goal of this study was to compare the properties of BK channels between PASMCs of normoxic (N) and chronic hypoxic (CH) rats and then explore key findings in human PASMCs. Polymerase chain reaction results revealed that 94.3% of transcripts encoding BKα pore proteins in PASMCs from N rats represent splice variants lacking the stress axis regulated exon insert, which confers oxygen sensitivity. Subsequent patch-clamp recordings from inside-out (I-O) patches confirmed a dense population of BK channels insensitive to hypoxia. The BK channels were highly activated by intracellular Ca2+ and the BKCO lithocholate; these responses require BKα-β1 subunit coupling. PASMCs of CH rats with established PH exhibited a profound overabundance of the dominant oxygen-insensitive BKα variant. Importantly, human BK (hBK) channels in PASMCs from human donor lungs also represented the oxygen-insensitive BKα variant activated by BKCOs. The hBK channels showed significantly enhanced Ca2+ sensitivity compared with rat BK channels. We conclude that rat BK and hBK channels in PASMCs are oxygen-insensitive BKα-β1 complexes highly sensitive to Ca2+ and the BKCO lithocholate. BK channels are overexpressed in PASMCs of a rat model of PH and may provide an abundant target for BKCOs designed to restore K+ efflux.

Abstract 055: Benefit of Mineralocorticoid Receptor Antagonism in Acute Kidney Injury: Role of Smooth Muscle Rac1

Introduction: Renal ischemia/reperfusion (IR) is a major cause of acute kidney injury (AKI). The benefit of novel non-steroidal MR antagonists such as finerenone in the IR context has not been evaluated and the mechanisms underlying the benefit of MR antagonism remain unclear. Objectives: To test the efficacy of finerenone in ischemic AKI and to evaluate the specific contribution of the MR expressed in endothelial or smooth muscle cell (SMC) in renal IR injury. Methods: We included 18 male C57/B6 mice that were divided in: sham, renal ischemia for 20 min and IR plus treatment with finerenone (10 mg/kg) by gavage once a day at -48, -24 and -1 h before IR. Alternatively, MR inactivation in endothelial cells (MR endoKO mice /Vecadh-cre) or in smooth muscle cells (MR SMCKO mice/SMA-cre) was induced in 3-month-old mice. Sham surgery or bilateral renal IR for 20 min was performed and mice were studied 24 h after reperfusion. Primary rat SMC cultures were used to assess the signaling pathways modulated by MR. Results: In C57/B6 WT, MR fl/fl and MR endoKO mice, IR induced kidney dysfunction and tubular injury. After IR, Finerenone-treated mice and the MR SMCKO mice presented normal renal function and a significant reduction of histological alterations, while MR endoKO mice were not protected. The benefit of finerenone and MR KO in SMC was associated with reduced oxidative stress-mediated lipid peroxidation as compared to MR fl/fl or WT mice. In aldosterone-stimulated rat SMC, we observed a 100% increase in hydrogen peroxide production and a 2-fold increase in Rac1 activity; MR and Rac1 antagonism blunted these effects. Moreover, mice deficient of Rac1 in SMC were also protected against ischemic AKI. Conclusion: Finerenone limits renal injury induced by IR. Moreover, genetic deletion of MR in SMC only has similar effects. This benefit was associated with reduced oxidative stress, by affecting oxidative stress production via SMC Rac1.

Hydrostatic pressure and muscarinic receptors are involved in the release of inflammatory cytokines in human bladder smooth muscle cells.

Abnormal intravesical pressure results in a series of pathological changes. We investigated the effects of hydrostatic pressure and muscarinic receptors on the release of inflammatory cytokines in rat and human bladder smooth muscle cells (HBSMCs). Animal model of bladder outlet obstruction was induced by urethra ligation. HBSMCs were subjected to elevated hydrostatic pressure and/or acetylcholine (Ach). Macrophage infiltration in the bladder wall was determined by immunohistochemical staining. The expression of inflammatory genes was measured by RT-PCR, ELISA and immunofluorescence. In obstructed bladder, inflammatory genes and macrophage infiltration were remarkably induced. When HBSMCs were subjected to 200-300 cm H2 O pressure for 2-24 h in vitro, the expressions of IL-6 and RANTES were significantly increased. Hydrostatic pressure promoted the protein levels of phospho-NFκB p65 and phospho-ERK1/2 as well as muscarinic receptors. Moreover, NFκB or ERK1/2 inhibitors suppressed pressure-induced inflammatory genes mRNA. When cells were treated with 1 μM acetylcholine for 6 h, a significant increase in IL-6 mRNA expression was detected. Acetylcholine also enhanced pressure-induced phospho-NFκB p65 and IL-6 protein expression. Additionally, pressure-induced IL-6 was partially suppressed by muscarinic receptors antagonists. Hydrostatic pressure and muscarinic receptors were involved in the secretion of inflammatory cytokines in HBSMCs, indicating a pro-inflammatory effect of the two factors in the pathological process of BOO.

Abstract 308: Differential Regulation of Alpha1a-adrenergic Receptor and its Genetic Variant Signaling by Novel Spinophilin-RGS2 Signalosome in Human Cells

α 1- adrenergic receptors (α 1a , α 1b , α 1d ), members of G protein-coupled receptor (GPCR) family, regulate stress-facilitated changes in the cardiovascular (CV) system and human blood pressure (BP) via smooth muscle cell (SMC) proliferation and vasoconstriction. The α 1a AR is the predominant subtype in human heart and resistance vessels. The α 1a AR G247R (247R) genetic variant was discovered in a patient with severe hypertension and in a cohort of patients enriched for CV disorders. We reported a unique coupling of this genetic variant to βarrestin/MMP/EGFR transactivation pathway in rat fibroblasts, cardiomyoblasts and vascular SMCs leading to hyperproliferation and hypertrophy. Here we present that differential interaction of 247R or α 1a AR (WT) with scaffolding/regulatory proteins mediate unique signaling of 247R. Physiological levels of 247R but not WT upregulate endogenous levels of scaffolding protein spinophilin (SPL) in rat SMCs and cardiomyoblasts. SPL knockdown or overexpression of RGS2 (negative R egulators of G -protein S ignaling) inhibits 247R-mediated hyperproliferation. Overexpression of SPL with RGS2 restores hyperproliferation suggesting that SPL binds RGS2 and prevents 247R-RGS2 interaction. To determine if 247R hyperproliferation is in effect in human cells, we generated human iPSCs from primary fibroblasts and differentiated them to SMCs with characteristic morphology and expression of SMC markers (RT-PCR at various passages). Consistent with our findings in rat cells, expression of 247R but not WT in hiPSC-derived SMCs triggered ~70% increased proliferation. Agonist treatment resulted in increased hypertrophy in 247R cells compared with WT. Co-immunoprecipitation from HEK293 cells expressing HA-α 1 ARs and full length Myc-SPL or Flag-RGS2 revealed that SPL exhibits ~2-fold stronger interaction with WT than with 247R. Consequently, SPL recruits RGS2 and inhibits receptor signaling. In contrast, weaker SPL-247R interaction results in diminished RGS2 inhibitory effect allowing hyperproliferation of 247R cells. Thus, we identify novel SPL/RGS2 signalosome responsible for α 1a AR signaling in human cells and identify SPL as a potential novel target for treatment of α 1 AR-mediated human CV disorders.

Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation

Phenotype switching of vascular smooth muscle cells (VSMC) from the contractile type to the synthetic type is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Inward rectifier K+ channel 2.1 (Kir2.1) has been identified in VSMC. However, whether it plays a functional role in regulating cellular transformation remains obscure. In this study, we evaluated the role of Kir2.1 on VSMC proliferation, migration, phenotype switching, and post-injury carotid neointimal formation. Kir2.1 knockdown significantly suppressed platelet-derived growth factor BB-stimulated rat vascular smooth muscle cells (rat-VSMC) proliferation and migration. Deficiency in Kir2.1 contributed to the restoration of smooth muscle α-actin, smooth muscle 22α, and calponin and to a reduction in osteopontin expression in rat-VSMC. Moreover, the in vivo study showed that rat-VSMC switched to proliferative phenotypes and that knockdown of Kir2.1 significantly inhibited neointimal formation after rat carotid injury. Kir2.1 may be a potential therapeutic target in the treatment of cardiovascular diseases, such as atherosclerosis and restenosis following percutaneous coronary intervention.

Changes in intracellular Ca2+ in the ureter smooth muscle cells of rats with neuropathic urinary tract dysfunction and its significance

Objective To investigate the changes in intracellular Ca2+ in the ureter smooth muscle cells (USMC) of rats with neuropathic urinary tract dysfunction (NUTD) and their significance. Methods Forty-five rats were randomly and averagely divided into NUTD group, experimental control (EC) group and blank control (BC) group.The NUTD group was operated with a spinal cord transection at the first lumbar level and the sacral cord was destroyed; in EC group the spinal process was partly bitten at the same position, but the spinal cord was not transected; BC group was given no operations.One week later, the video-urodynamic was performed to observe the acontractile detrusor (ACD), vesicoureteral reflux (VUR) and urinary tract dysfunction in rats among the NUTD group, EC group and the BC group.Video-urodynamic assessment was performed at the sixth week after operation.Ureter smooth muscle cells (USMC) were obtained by collagenase digestion.Intracellular Ca2+ in the USMC were observed by laser scanning confocal microscope.Then the effects of Bay K8644(10-8 mol/L, 10-7 mol/L, 10-6 mol/L) on cytosolic Ca2+ concentrations( [Ca2+ ]i) in NUTD group were studied by calculating the fluorescence intensity. Results ACD and no detrusor overactivity were found in all rats in NUTD group and without vesicoureteral reflux.Immunofluorescence method confirmed that the cells were USMC.Compared with BC group(31.44±2.82)and EC group(32.06±3.67), the fluorescence intensity(FI) of intracellular Ca2+ in USMC was much lower in the NUTD group(9.80±1.11), and there was significant difference(P<0.05). Bay K8644 (10-8mol/L, 10-7mol/L, 10-6mol/L) increased the FI of [Ca2+ ]I in a concentration-dependent manner, which were 3.80±1.30, 10.04±2.15, 19.89±2.06, respectively, and there was significant difference (P<0.05). Conclusions The decrease in Ca2+ concentration in the ureter smooth muscle cells may be one of the important factors for the primary ureteral dysfunction of NUTD.And calcium channel agonist can be meaningful for adjusting abnormal Ca2+ concentration in USMC of NUTD. Key words: Neurogenic urinary tract dysfunction; Ureter smooth muscle cell; L-type Ca2+ channel; Bay K8644; Laser scanning confocal microscope