Douglas K. Bowles

Increased expression of K_Ca3.1 has been found in vascular smooth muscle cells (SMC), macrophages, and T cells in atherosclerotic lesions from humans and mice. Pharmacological inhibition of K_Ca3.1 in limiting atherosclerosis has been demonstrated in mice and pigs, however direct, loss-of-function, i.e. gene silencing, studies are absent. Therefore, we generated K_Ca3.1^-/-Apoe^-/- (DKO) mice and assessed lesion development in the brachiocephalic artery (BCA) of DKO versus Apoe^-/- mice on a Western diet for 3 months. In BCAs of DKO mice, lesion size and relative stenosis were reduced by ~70% compared to Apoe^-/- mice, with no effect on medial or lumen area. Additionally, DKO mice exhibited a significant reduction in macrophage content within plaques compared to Apoe^-/- mice, independent of sex. In vitro migration assays showed a significant reduction in migration of bone marrow-derived macrophages (BMDMs) from DKO mice compared to those from Apoe^-/- mice. In vitro experiments using rat aortic smooth muscle cells revealed inhibition of PDGF-BB-induced MCP1/Ccl2 expression upon K_Ca3.1 inhibition, while activation of K_Ca3.1 further enhanced MCP1/Ccl2 expression. Both in vivo and in vitro analyses showed that silencing K_Ca3.1 had no significant effect on the collagen content of plaque. RNAseq analysis of BCA samples from DKO and Apoe^-/- mice revealed PPAR-dependent signaling as a potential key mediator of the reduction in atherosclerosis due to K_Ca3.1 silencing. Overall, this study provides the first genetic evidence that K_Ca3.1 is a critical regulator of atherosclerotic lesion development and composition and provides novel mechanistic insight into the link between K_Ca3.1 and atherosclerosis.

Fibrosis represents a pivotal pathological process in numerous diseases, characterized by excessive deposition of extracellular matrix (ECM) that disrupts normal tissue architecture and function. In the heart, cardiac fibrosis significantly impairs both structural integrity and functional capacity, contributing to the progression of heart failure. Central to this process are cardiac fibroblasts (CFs), which, upon activation, differentiate into contractile myofibroblasts, driving pathological ECM accumulation. Transforming growth factor-beta (TGFβ) is a well-established regulator of fibroblast activation; however, the precise molecular mechanisms, particularly the involvement of ion channels, remain poorly understood. Emerging evidence highlights the regulatory role of ion channels, including calcium-activated potassium (K_Ca) channels, in fibroblast activation. This study elucidates the role of ion channels and investigates the mechanism by which Yoda1, an agonist of the mechanosensitive ion channel Piezo1, modulates TGFβ-induced fibroblast activation. Using NIH/3T3 fibroblasts, we demonstrated that TGFβ-induced activation is regulated by tetraethylammonium (TEA)-sensitive potassium channels, but not by specific K⁺ channel subtypes such as BK, SK, or IK channels. Intriguingly, Yoda1 was found to inhibit TGFβ-induced fibroblast activation through a Piezo1-independent mechanism. Transcriptomic analysis revealed that Yoda1 modulates fibroblast activation by altering gene expression pathways associated with fibrotic processes. Bromodomain-containing protein 4 (BRD4) was identified as a critical mediator of Yoda1’s effects, as pharmacological inhibition of BRD4 with JQ1 or ZL0454 suppressed TGFβ-induced expression of the fibroblast activation marker Periostin (Postn). Conversely, BRD4 overexpression attenuated the inhibitory effects of Yoda1 in both mouse and rat CFs. These results provide novel insights into the pharmacological modulation of TGFβ-induced cardiac fibroblast activation and highlight promising therapeutic targets for the treatment of fibrosis-related cardiac pathologies.

Increased expression of K _Ca 3.1 has been found in vascular smooth muscle (SMC), macrophages, and T cells in atherosclerotic lesions from humans and mice. Proliferating SMC cells increase the expression of K _Ca 3.1, such that it becomes a dominant K ⁺ channel and contributes to SMC cell migration. The efficacy of pharmacological inhibition of K _Ca 3.1 in limiting atherosclerosis progression has been demonstrated in mice and pigs, however direct, loss-of-function, i.e. gene silencing, studies are absent. To investigate the role of K _Ca 3.1, we used CRISPR/Cas9 to generate K _Ca 3.1 ^-/- Apoe ^-/- (DKO) mice and assessed lesion development in the brachiocephalic artery (BCA) of DKO versus Apoe ^-/- mice on a Western diet for 3 months. Notably, the loss of K _Ca 3.1 did not affect serum total cholesterol or body weight. In BCAs of DKO mice, lesion size (0.036 mm² vs. 0.118 mm², p-/- mice, with no effect on medial or lumen area. Additionally, DKO mice exhibited a significant reduction in macrophage content within atherosclerotic plaques compared to Apoe ^-/- mice, independent of sex. In vitro migration assays further showed a significant reduction in migration of bone marrow-derived macrophages (BMDMs) from DKO mice compared to those from Apoe ^-/- mice. Furthermore, in vitro experiments using rat aortic smooth muscle cells (RAOSMCs) revealed significant inhibition of PDGF-BB-induced MCP1/Ccl21 expression upon K _Ca 3.1 inhibition, while activation of K _Ca 3.1 further enhanced MCP1/Ccl21 expression. Both in vivo and in vitro analyses showed that silencing K _Ca 3.1 and sex had no significant effect on the collagen content of plaque. RNAseq analysis of BCA samples from DKO and Apoe ^-/- mice revealed PPAR-dependent signaling as a potential key mediator of the reduction in atherosclerosis due to K _Ca 3.1 silencing. Overall, this study provides the first genetic evidence that K _Ca 3.1 is a critical regulator of atherosclerotic lesion development and composition and provides novel mechanistic insight into the link between K _Ca 3.1 and atherosclerosis.

The objective of this study was to evaluate the biocompatibility of trimethylsilane (TMS) plasma nanocoatings modified with NH₃/O₂ (2:1 molar ratio) plasma post-treatment onto cobalt chromium (CoCr) L605 alloy coupons and stents for cardiovascular stent applications. Biocompatibility of plasma nanocoatings was evaluated by coating adhesion, corrosion behavior, ion releasing, cytotoxicity, and cell proliferation. Surface chemistry and wettability were studied to understand effects of surface properties on biocompatibility. Results show that NH₃/O₂ post-treated TMS plasma nanocoatings are hydrophilic with water contact angle of 48.5° and have a typical surface composition of O (39.39 at.%), Si (31.92 at.%), C (24.12 at.%), and N (2.77 at.%). The plasma nanocoatings were conformal to substrate surface topography and had excellent adhesion to the alloy substrates, as assessed by tape test (ASTM D3359), and showed no cracking or peeling off L605 stent surfaces after dilation. The plasma nanocoatings also improve the corrosion resistance of CoCr L605 alloy by increasing corrosion potential and decreasing corrosion rates with no pitting corrosion and no mineral adsorption layer. Ion releasing test revealed that Co, Cr, and Ni ion concentrations were reduced by 64–79%, 67–69%, and 57–72%, respectively, in the plasma-nanocoated L605 samples as compared to uncoated L605 control samples. The plasma nanocoatings showed no sign of cytotoxicity from the test results according to ISO 10993-05 and 10993-12. Seven-day cell culture demonstrated that, in comparison with the uncoated L605 control surfaces, the plasma nanocoating surfaces showed 62 ± 7.3% decrease in porcine coronary artery smooth muscle cells (PCASMCs) density and had comparable density of porcine coronary artery endothelial cells (PCAECs). These results suggest that TMS plasma nanocoatings with NH₃/O₂ plasma post-treatment possess the desired biocompatibility for stent applications and support the hypothesis that nanocoated stents could be very effective for in-stent restenosis prevention.

Low, oscillatory flow/shear patterns are associated with atherosclerotic lesion development. Increased expression of K_Ca3.1 has been found in Vascular Smooth Muscle (VSM), macrophages and T-cells in lesions from humans and mice. Increased expression of K_Ca3.1, is also required for VSM cell proliferation and migration. Previously, we showed that the specific K_Ca3.1 inhibitor, TRAM-34, could inhibit coronary neointimal development following balloon injury in swine. Atherosclerosis develops in regions with a low, oscillatory (i.e. atheroprone) flow pattern. Therefore, we used the Partial Carotid Ligation (PCL) model in high-fat fed, Apoe^−/− mice to determine the role of K_Ca3.1 in atherosclerotic lesion composition and development. PCL was performed on 8–10 week old male Apoe^−/− mice and subsequently placed on a Western diet (TD.88137, Teklad) for 4 weeks. Mice received daily s.c. injections of TRAM-34 (120 mg/kg) or equal volumes of vehicle (peanut oil, PO). 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) treatment reduced lesion size ~50% (p < 0.05). In addition, lesions from TRAM-34 treated mice contained less collagen (6% ± 1% vs. 15% ± 2%; p < 0.05), fibronectin (14% ± 3% vs. 32% ± 3%; p < 0.05) and smooth muscle content (19% ± 2% vs. 29% ± 3%; p < 0.05). Conversely, TRAM-34 had no effect on total cholesterol (1455 vs. 1334 mg/dl, PO and TRAM, resp.) or body weight (29.1 vs. 28.8 g, PO and TRAM, resp.). Medial smooth muscle of atherosclerotic carotids showed diminished RE1-Silencing Transcription Factor (REST)/Neural Restrictive Silencing Factor (NRSF) expression, while REST overexpression in vitro inhibited smooth muscle migration. Together, these data support a downregulation of REST/NRSF and upregulation of K_Ca3.1 in determining smooth muscle and matrix content of atherosclerotic lesions.

Human studies demonstrate that physical activity reduces both morbidity and mortality of coronary heart disease (CHD) including decreased progression and/or regression of CHD with life‐style modification which includes exercise. However, evidence supporting an intrinsic, direct effect of exercise in attenuating the development of CHD is equivocal. One limitation has been the lack of a large animal model with clinically evident CHD disease. Thus, we examined the role of endurance exercise in CHD development in a swine model of familial hypercholesterolemia (FH) that exhibits robust, complex atherosclerosis. FH swine were randomly assigned to either sedentary (Sed) or exercise trained (Ex) groups. At 10 months of age, Ex pigs began a 10 months, moderate‐intensity treadmill‐training intervention. At 14 months, all pigs were switched to a high‐fat, high‐cholesterol diet. CHD was assessed by intravascular ultrasound (IVUS) both prior to and after completion of 6 months on the HFC diet. Prior to HFC diet, Ex resulted in a greater coronary artery size in the proximal and mid sections of the LCX compared to SED, with no effect in the LAD. After 6 months on HFC diet, there was a 5–6 fold increase in absolute plaque volume in all segments of the LCX and LAD in both groups. At 20 months, there was no difference in vessel volume, lumen volume, absolute or relative plaque volume in either the LCX or LAD between Sed and Ex animals. These findings fail to support an independent, direct effect of exercise in limiting CHD progression in familial hypercholesterolemia.

NADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca²⁺-activated K⁺ channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors. Dihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration. Our findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis.

Objective Sex hormone status has been demonstrated to play a role in the regulation of ion channel activity. We previously demonstrated increased L-type Ca²⁺ channel current (I_Ca) in the coronary smooth muscle cells (SMCs) of male swine compared with female swine. In male swine, endogenous testosterone increases I_Ca in SMCs by enhanced expression of the pore-forming α1 subunit Ca_v1.2. Conversely, the role of sex hormones in female swine has not previously been investigated. Therefore, the purpose of the current study was to determine the effect of ovariectomy (OVX) on L-type Ca²⁺ channel activity and expression in female Yucatan miniature swine. Methods Sexually mature female swine were obtained from a breeder and either left intact (intact female [IF]; n = 5) or ovariectomized (n = 6). Results Sensitivity to depolarization-induced contractions was increased by OVX. Accordingly, mean (SEM) I_Ca was enhanced in the OVX group (−9.5 [0.6] pA/pF) compared with the IF group (−4.5 [0.3] pA/pF), although L-type Ca²⁺ channel α1 subunit (Ca_v1.2; α1c) messenger RNA (mRNA) and protein expressions were unchanged. Among the L-type Ca²⁺ channel β subunits, β1 (188 [31]) and β2a (561 [79]) had higher mRNA expression levels (target/18S) than β3 (9 [1]) and β4 (2 [0.1]). Although β2a, β3, and β4 mRNA and protein expressions were not different between groups, protein expression of the β1 subunit (Ca_vβ1) was decreased in the OVX group compared with the IF group. Conclusions Endogenous female hormones inhibit L-type Ca²⁺ channel activity in coronary SMCs potentially via the up-regulation of Ca_vβ1 subunit expression.

Background: Low endogenous testosterone levels have been shown to be a risk factor for the development of cardiovascular disease and cardiovascular benefits associated with testosterone replacement therapy are being advocated; however, the effects of endogenous testosterone levels on acute coronary vasomotor responses to androgen administration are not clear. The objective of this study was to compare the effects of acute androgen administration onin vivocoronary conductance andin vitrocoronary microvascular diameter in intact and castrated male swine.Methods: Pigs received intracoronary infusions of physiologic levels (1–100 nM) of testosterone, the metabolite 5α-dihydrotestosterone, and the epimer epitestosterone while left anterior descending coronary blood flow and mean arterial pressure were continuously monitored. Following sacrifice, coronary arterioles were isolated, cannulated, and exposed to physiologic concentrations (1–100 nM) of testosterone, 5α-dihydrotestosterone, and epitestosterone. To evaluate effects of the androgen receptor on acute androgen dilation responses, real-time PCR and immunohistochemistry for androgen receptor were performed on conduit and resistance coronary vessels.Results: In vivo, testosterone and 5α-dihydrotestosterone produced greater increases in coronary conductance in the intact compared to the castrated males.In vitro, percent maximal dilation of microvessels was similar between intact and castrated males for testosterone and 5α-dihydrotestosterone. In both studies epitestosterone produced significant increases in conductance and microvessel diameter from baseline in the intact males. Androgen receptor mRNA expression and immunohistochemical staining were similar in intact and castrated males.Conclusions: Acute coronary vascular responses to exogenous androgen administration are increased by endogenous testosterone, an effect unrelated to changes in androgen receptor expression.