Increased expression of KCa3.1 has been found in vascular smooth muscle cells (SMC), macrophages, and T cells in atherosclerotic lesions from humans and mice. Pharmacological inhibition of KCa3.1 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 KCa3.1-/-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 KCa3.1 inhibition, while activation of KCa3.1 further enhanced MCP1/Ccl2 expression. Both in vivo and in vitro analyses showed that silencing KCa3.1 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 KCa3.1 silencing. Overall, this study provides the first genetic evidence that KCa3.1 is a critical regulator of atherosclerotic lesion development and composition and provides novel mechanistic insight into the link between KCa3.1 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 (KCa) 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.

Although murine models of coronary atherosclerotic disease have been used extensively to determine mechanisms, limited new therapeutic options have emerged. Pigs with familial hypercholesterolemia (FH pigs) develop complex coronary atheromas that are almost identical to human lesions. We reported previously that insulin-like growth factor 1 (IGF-1) reduced aortic atherosclerosis and promoted features of stable plaque in a murine model. We administered human recombinant IGF-1 or saline (control) in atherosclerotic FH pigs for 6 months. IGF-1 decreased relative coronary atheroma in vivo (intravascular ultrasound) and reduced lesion cross-sectional area (postmortem histology). IGF-1 increased plaque's fibrous cap thickness, and reduced necrotic core, macrophage content, and cell apoptosis, consistent with promotion of a stable plaque phenotype. IGF-1 reduced circulating triglycerides, markers of systemic oxidative stress, and CXCL12 chemokine levels. We used spatial transcriptomics (ST) to identify global transcriptome changes in advanced plaque compartments and to obtain mechanistic insights into IGF-1 effects. ST analysis showed that IGF-1 suppressed FOS/FOSB factors and gene expression of MMP9 and CXCL14 in plaque macrophages, suggesting possible involvement of these molecules in IGF-1's effect on atherosclerosis. Thus, IGF-1 reduced coronary plaque burden and promoted features of stable plaque in a pig model, providing support for consideration of clinical trials.

Background: Macrophage activation and vascular inflammation play important roles in atherosclerosis. Non-editing functions of adenosine deaminase acting on RNA (ADAR1) in macrophage activation and atherosclerosis remain elusive. Methods: Nonvascular ADAR1 in atherosclerosis progression was investigated via aortic transplantation and bone marrow transplantation. ApoE-/- mice combined with macrophage-specific ADAR1 deficiency were used to determine macrophage-specific roles of ADAR1 in atherosclerosis. Human coronary atherosclerotic specimens were utilized to establish the relevance to human atherosclerosis. Moreover, a humanized atherosclerosis model was created to examine the influence of human macrophages in human arteries. Results: ADAR1+/– attenuates atherosclerosis. Allograft transplantation of wild-type abdominal aorta into ADAR1+/– recipient mice promoted atherosclerosis. Likewise, bone marrow transplantation from wild-type mice to ADAR1+/– recipient mice negated the protective effects of ADAR1+/–, suggesting that nonvascular ADAR1 is instrumental for atherosclerosis progression. ADAR1 deficiency in hematopoietic cells lessened the prevalence and severity of atherosclerosis, and impeded macrophage infiltration, foam cell formation, and aortic wall inflammation. Mechanistically, ADAR1 deletion deterred classical macrophage activation and foam cell formation via downregulation of PPAR-γ, which was mediated by enhancing ubiquitination and further degradation of PPAR-γ. Importantly, ADAR1 was upregulated in macrophages in human atherosclerotic lesions. Transplantation of ADAR1-deficient human monocytes, rather than their wild-type counterparts, into hyperlipidemic immunodeficient mice suppressed the formation of atherosclerosis in transplanted arteries from patients undergoing coronary artery bypass grafting surgery due to coronary artery blockade by atherosclerotic plagues. Interestingly, ADAR1 suppression in macrophages noticeably augmented the anti-atherosclerotic effect of the PPAR-γ agonist rosiglitazone. Conclusions: These results demonstrate that noncanonical ADAR1 plays an essential role in the regulation of macrophage activation and atherosclerosis development.

In-stent restenosis and thrombosis remain to be long-term challenges in coronary stenting procedures. The objective of this study was to evaluate the in vitro biological responses of trimethylsilane (TMS) plasma nanocoatings modified with NH3 /O2 (2:1 molar ratio) plasma post-treatment (TMS + NH3 /O2 nanocoatings) on cobalt chromium (CoCr) alloy L605 coupons, L605 stents, and 316L stainless steel (SS) stents. Surface properties of the plasma nanocoatings with up to 2-year aging time were characterized by wettability assessment and x-ray photoelectron spectroscopy (XPS). It was found that TMS + NH3 /O2 nanocoatings had a surface composition of 41.21 ± 1.06 at% oxygen, 31.90 ± 1.08 at% silicon, and 24.12 ± 1.7 at% carbon, and very small but essential amount of 2.77 ± 0.18 at% nitrogen. Surface chemical stability of the plasma coatings was noted with persistent O/Si atomic ratio of 1.292-1.413 and N/Si atomic ratio of ~0.087 through 2 years. The in vitro biological responses of plasma nanocoatings were studied by evaluating the cell proliferation and migration of porcine coronary artery endothelial cells (PCAECs) and smooth muscle cells (PCASMCs). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay results revealed that, after 7-day incubation, TMS + NH3 /O2 nanocoatings maintained a similar level of PCAEC proliferation while showing a decrease in the viability of PCASMCs by 73 ± 19% as compared with uncoated L605 surfaces. Cell co-culture of PCAECs and PCASMCs results showed that, the cell ratio of PCAEC/PCASMC on TMS + NH3 /O2 nanocoating surfaces was 1.5-fold higher than that on uncoated L605 surfaces, indicating enhanced selectivity for promoting PCAEC growth. Migration test showed comparable PCAEC migration distance for uncoated L605 and TMS + NH3 /O2 nanocoatings. In contrast, PCASMC migration distance was reduced nearly 8.5-fold on TMS + NH3 /O2 nanocoating surfaces as compared to the uncoated L605 surfaces. Platelet adhesion test using porcine whole blood showed lower adhered platelets distribution (by 70 ± 16%), reduced clotting attachment (by 54 ± 12%), and less platelet activation on TMS + NH3 /O2 nanocoating surfaces as compared with the uncoated L605 controls. It was further found that, under shear stress conditions of simulated blood flow, TMS + NH3 /O2 nanocoating significantly inhibited platelet adhesion compared to the uncoated 316L SS stents and TMS nanocoated 316L SS stents. These results indicate that TMS + NH3 /O2 nanocoatings are very promising in preventing both restenosis and thrombosis for coronary stent applications.

Introduction: Cardiovascular disease, including atherosclerosis is the leading cause of mortality in the Western world. We have reported that insulin-like growth factor I (IGF-1) reduced atherosclerosis in the murine model, however it is unknown whether IGF-1 exerts protective effect on human-like atherosclerosis. Hypothesis: Rapacz pigs with familial hypercholesterolemia (FH pigs) fed with high-cholesterol diet develop human-like advanced plaques in coronary arteries. We hypothesize that IGF-1 suppresses atherosclerosis in FH pigs. Methods: Human recombinant IGF-1 ( 50 ug/kg) or saline (control) was injected daily into high-fat diet fed FH swine (n=9/group) for 6 months. Atherosclerotic burden was quantified by intravascular ultrasound (IVUS) at basal level (T0), after 3 months on diet (T3) and at sacrificing (T6). Results: IGF-1-injected pigs had 2.6-fold increase in total plasma IGF-1 (ELISA, p<0.001 vs. control pigs). Pigs in IGF-1 and control groups had a gradual increase in vessel volume in the right coronary artery (RCA), and left anterior descending artery (LAD) with time as quantified by IVUS. However, IGF-1-injected pigs had a larger increase in vessel volume after 3 and 6 months compared to controls suggesting vascular hypertrophy. The atherosclerotic burden was increased in RCA, and LAD in both groups after 3 and 6 months of injections compared to the pre-injection time point. We found a significant reduction in atherosclerotic burden in RCA and LAD in IGF-1 group compared to control at 6 months’ time-point (RCA: IGF-1, 203±13%, vs. control, 244±8; LAD: IGF-1, 200±6%, vs. control, 228±11%). The lumen volume of coronaries was decreased in both groups, however, coronaries in IGF-1-injected pigs had on average 24±3% larger lumen volume compared to control pigs at 6 months’ time-point. Conclusions: We found that IGF-1 increased total volume of pig coronaries suggesting hypertrophy and these data are consistent with growth-stimulating effects of IGF-1. We found reduction in atherosclerotic burden and increase in lumen volume in coronaries in the IGF-1 group after 6 months of injections consistent with an anti-atherosclerotic effect of IGF-1. Our data support the feasibility of using IGF-1 for as an anti-atherogenic therapy.

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.

Impaired coronary microvascular function (e.g., reduced dilation and coronary flow reserve) predicts cardiac mortality in obesity, yet underlying mechanisms and potential therapeutic strategies remain poorly understood. Mineralocorticoid receptor (MR) antagonism improves coronary microvascular function in obese humans and animals. Whether MR blockade improves in vivo regulation of coronary flow, a process involving voltage-dependent K+ (Kv) channel activation, or reduces coronary structural remodeling in obesity is unclear. Thus, the goals of this investigation were to determine the effects of obesity on coronary responsiveness to reductions in arterial PO2 and potential involvement of Kv channels and whether the benefit of MR blockade involves improved coronary Kv function or altered passive structural properties of the coronary microcirculation. Hypoxemia increased coronary blood flow similarly in lean and obese swine; however, baseline coronary vascular resistance was significantly higher in obese swine. Inhibition of Kv channels reduced coronary blood flow and augmented coronary resistance under baseline conditions in lean but not obese swine and had no impact on hypoxemic coronary vasodilation. Chronic MR inhibition in obese swine normalized baseline coronary resistance, did not influence hypoxemic coronary vasodilation, and did not restore coronary Kv function (assessed in vivo, ex vivo, and via patch clamping). Lastly, MR blockade prevented obesity-associated coronary arteriolar stiffening independent of cardiac capillary density and changes in cardiac function. These data indicate that chronic MR inhibition prevents increased coronary resistance in obesity independent of Kv channel function and is associated with mitigation of obesity-mediated coronary arteriolar stiffening.

Prospective, cohort studies in humans demonstrate that moderate to high levels of physical activity reduce both morbidity and mortality of coronary heart disease (CHD) including a decreased progression and/or regression of CHD with life‐style modification which includes exercise. However, a thorough review of both human and animal literature reveals equivocal evidence to support an intrinsic, direct exercise effect in attenuating the development of CHD. Exercise reduces development and/or causes regression of atherosclerotic lesions in mice and rabbits, however in larger mammals including primates, the evidence is more equivocal. A major limitation has been the lack of large animal models with clinically evident CHD disease. Thus, we sought to determine the effect of endurance exercise in CHD development and compensatory remodeling in a swine model of familial hypercholesterolemia (FH) that exhibits robust, complex atherosclerosis. Castrated male Rapacz familial hypercholesterolemic (FH) swine were obtained from the University of Wisconsin Swine Research and Teaching Center. Pigs were randomly assigned to either sedentary (Sed; n=9) or exercise trained (Ex; n=8) groups. At 10 months of age, Ex pigs began a 10 month treadmill‐training intervention consisting of moderate‐intensity (70% of maximal heart rate) once per day, for 5 days each week. At 14 months, all pigs were switched to a high‐fat, high‐cholesterol diet (HF; by weight 13% protein, 21.3% fat, 41.4% carbohydrate, 2% cholesterol and 1% sodium cholate). CHD was assessed by intravascular ultrasound (IVUS) both prior to (14 months of age) and after completion of 6 months on the HF diet (20 months of age). Angiograms and IVUS were obtained using standard coronary catheterization techniques. IVUS pullbacks (0.5 mm/sec; Galaxy II, Boston Scientific, 40MHz) were obtained for the proximal anterior descending (LAD) and left circumflex (LCX) arteries. For analysis, the total segment was subdivided into 3 sections and total vessel volume, lumen volume, total plaque burden volume and percent plaque burden were determined in the proximal, mid, and distal segments from 3D reconstruction (QIVUS software; Medis). Prior to HF diet, Ex resulted in a greater coronary artery size (vessel and lumen volume) in the proximal and mid sections of the LCX compared to SED, with no effect in the LAD. Relative plaque volume was minimal (~15–20% vessel area) in both Sed and Ex groups. After 6 months on HF diet, there was a 5–6 fold increase in absolute plaque volume in all segments of the LCX and LAD in both groups. Ex had no effect on absolute plaque volume at 20 months in any segment of either LCX or LAD. In both Sed and Ex groups, vessel volume was greater at 20 months compared to 14 months such that lumen volume was maintained despite the increase in plaque volume, i.e. compensatory outward remodeling was similar in both groups. Overall at 20 months, there was no significant 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.Support or Funding InformationNIH HL52490This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effects of oleanolic acid (OA) on the fertility of male mice were investigated using both invivo and invitro experimental models. The experimental group (n=12) was treated with a daily dose of 30mgOAkg-1 bodyweight (i.p.), while the control group (n=6) received a daily dose of 10% ethanol solution (1mLkg-1 bodyweight). The effect of OA on the permeability status of TM4 Sertoli monolayers was investigated by measuring the transepithelial electrical resistance (TER), intracellular electrical resistance and semiquantitative RT-PCR. After 45 days, OA-treated males produced no pregnancies but in the control group, all 12 females were impregnated (69 offspring). Male mice, which demonstrated sterility when exposed to OA, recovered their fertility after 30 days (78 offspring). Testicular histological observations of OA-treated mice showed detachment of adjacent Sertoli-Sertoli cells. A control monolayer developed TER of 300-400 Ω.cm2, but OA (50, 100, 200µgL-1) treated monolayers developed TER of approximately 100Ω.cm2. Intracellular electrophysiological and RT-PCR data supported the premise that OA compromised tight junctional permeability. The study demonstrated reversible contraception in male mice by increasing the permeability of the germinal epithelium and further postulates that contraceptive reversibility is brought about by the reconstitution of the paracellular junctions between adjacent Sertoli cells.

Increased expression of KCa3.1 has been found in vascular smooth muscle (VSM), macrophages and T cells in atherosclerotic lesions from humans and mice. Proliferating VSM cells increase expression of KCa3.1, such that it becomes a dominant K+ channel and contributes to VSM cell migration. Previously, we showed that the KCa3.1 inhibitor, TRAM‐34, could inhibit coronary neointimal development following coronary balloon injury in swine. In the current study, we tested the role of KCa3.1 in atherosclerotic lesion development using two mouse models of atherosclerosis. First, partial carotid ligation (PCL), which produces a low, oscillatory (i.e. atheroprone) flow pattern in the left carotid artery was performed on 6–8 week old male Apoe−/− mice. Mice were subsequently placed on a Western diet (WD; TD.88137, Teklad) for 4 weeks and received daily s.c. injections of TRAM‐34 (120 mg/kg) or equal volumes of vehicle (peanut oil). To directly test the role of KCa3.1 we used CRISPR/Cas9 to generate KCa3.1−/−Apoe−/− (DKO) mice. Subsequently, we examined lesion development in the brachiocephalic artery (BCA) of DKO vs. Apoe−/− male mice on a WD for 3 months. In PCL mice, TRAM‐34 treatment reduced lesion size ~50% (p<0.05). In addition, lesions from TRAM‐34 treated mice contained significantly less collagen (6 ± 1% v. 15 ± 2%; p<0.05), fibronectin (14±3% v. 32±3%; p<0.05) and smooth muscle content (19±2% v. 29±3%; p<0.05). Similarly, in BCAs of DKO mice, both lesion size (0.036 vs. 0.118 mm2, p<0.05) and relative stenosis (13.9% vs. 43.0%, p<0.05) were reduced 70% compared to Apoe−/− mice with no effect on medial or lumen area. Neither TRAM‐34 nor KCa3.1 silencing had any effect on total cholesterol or body weight. These data provide the first genetic validation defining a major role of KCa3.1 in lesion development and determining smooth muscle and matrix content of atherosclerotic lesions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

OBJECTIVE:Swine with familial hypercholesterolemia (FH) exhibit attenuated exercise-induced systemic vasodilation that is restored by phosphodiesterase 5 (PDE5) inhibition. Whether the impacts of FH and PDE5 inhibition to impair and restore exercise-induced vasodilation, respectively, results from tissue-specific or generalized effects remains unclear. Thus, we hypothesized that FH induces generalized impairment of skeletal muscle vasodilation that would be alleviated by PDE5 inhibition.METHODS:Systemic vascular responses to exercise were assessed in chronically instrumented normal and FH swine before and after PDE5 inhibition with EMD360527. Skeletal muscle and organ blood flows and conductances were determined via the microsphere technique.RESULTS:As previously reported, vs normal swine, FH swine have pronounced elevation of total cholesterol and impaired exercise-induced vasodilation that is restored by PDE5 inhibition. Blood flows to several, not all, skeletal muscle vascular beds were severely impaired by FH associated with reduced blood flow to many visceral organs. PDE5 inhibition differentially impacted skeletal muscle and organ blood flows in normal and FH swine.CONCLUSIONS:These data indicate that FH induces regional, not generalized, vasomotor dysfunction and that FH and normal swine exhibit unique tissue blood flow responses to PDE5 inhibition thereby adding to accumulating evidence of vascular bed-specific dysfunction in co-morbid conditions.

Elevated plasma aldosterone (Aldo) levels are associated with greater risk of cardiac ischemic events and cardiovascular mortality. Adenosine-mediated coronary vasodilation is a critical cardioprotective mechanism during ischemia; however, whether this response is impaired by increased Aldo is unclear. We hypothesized that chronic Aldo impairs coronary adenosine-mediated vasodilation via downregulation of vascular K+ channels. Male C57BL/6J mice were treated with vehicle (Con) or subpressor Aldo for 4 wk. Coronary artery function, assessed by wire myography, revealed Aldo-induced reductions in vasodilation to adenosine and the endothelium-dependent vasodilator acetylcholine but not to the nitric oxide donor sodium nitroprusside. Coronary vasoconstriction to endothelin-1 and the thromboxane A(2) mimetic U-46619 was unchanged by Aldo. Additional mechanistic studies revealed impaired adenosine A(2A), not A(2B), receptor-dependent vasodilation by Aldo with a tendency for Aldo-induced reduction of coronary A(2A) gene expression. Adenylate cyclase inhibition attenuated coronary adenosine dilation but did not eliminate group differences, and adenosine-stimulated vascular cAMP production was similar between Con and Aldo mice. Similarly, blockade of inward rectifier K+ channels reduced but did not eliminate group differences in adenosine dilation whereas group differences were eliminated by blockade of Ca2+-activated K+ (K-Ca) channels that blunted and abrogated adenosine and A(2A)-dependent dilation, respectively. Gene expression of several coronary K-Ca channels was reduced by Aldo. Together, these data demonstrate Aldo-induced impairment of adenosine-mediated coronary vasodilation involving blunted A(2A)-K-Ca-dependent vasodilation, independent of blood pressure, providing important insights into the link between plasma Aldo and cardiac mortality and rationale for aldosterone antagonist use to preserve coronary microvascular function. NEW & NOTEWORTHY Increased plasma aldosterone levels are associated with worsened cardiac outcomes in diverse patient groups by unclear mechanisms. We identified that, in male mice, elevated aldosterone impairs coronary adenosine-mediated vasodilation, an important cardioprotective mechanism. This aldosterone-induced impairment in-volves reduced adenosine A(2A), not A(2B), receptor-dependent vasodilation associated with downregulation of coronary K-Ca channels and does not involve altered adenylate cyclase/cAMP signaling. Importantly, this effect of aldosterone occurred independent of changes in coronary vasoconstrictor responsiveness and blood pressure.

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo.

EXT‐induced arteriolar adaptations in skeletal muscle are heterogeneous because of spatial variations in muscle fiber type composition and fiber recruitment patterns during exercise. The purpose of this report is to summarize a series of experiments conducted to test the hypothesis that changes in vascular gene expression are signaled by alterations in shear stress resulting from increases in blood flow, muscle fiber type composition, and fiber recruitment patterns. We also report results from a follow‐up study of Ankrd23, one gene whose expression was changed by EXT. We expected to see differences in magnitude of changes in gene expression along arteriolar trees and between/among arteriolar trees but similar directional changes. However, transcriptional profiles of arterioles/arteries from OLETF rats exposed to END or SIT reveal that EXT does not lead to similar directional changes in the transcriptome among arteriolar trees of different skeletal muscles or along arteriolar trees within a particular muscle. END caused the most changes in gene expression in 2A arterioles of soleus and white gastrocnemius with little to no changes in the FAs. Ingenuity Pathway Analysis across vessels revealed significant changes in gene expression in 18 pathways. EXT increased expression of some genes (Shc1, desert hedgehog protein (Dhh), adenylate cyclase 4 (Adcy4), G protein‐binding protein, alpha (Gnat1), and Bcl2l1) in all arterioles examined, but decreased expression of ubiquitin D (Ubd) and cAMP response element modulator (Crem). Many contractile and/or structural protein genes were increased by SIT in the gastrocnemius FA, but the same genes exhibited decreased expression in red gastrocnemius arterioles. Ankrd23 mRNA levels increased with increasing branch order in the gastrocnemius arteriolar tree and were increased 19‐fold in gastrocnemius muscle FA by SIT. Follow‐up experiments indicate that Ankrd23 mRNA level was increased 14‐fold in cannulated gastrocnemius FA when intraluminal pressure was increased from 90 and 180 cm H2O for 4 hours. Also, Ankrd23−/− mice exhibit limited ability to form collateral arteries following femoral artery occlusion compared to WT mice (angioscore WT=0.18±0.03; Ankrd23−/−=0.04±0.01). Further research will be required to determine whether Ankrd23 plays an important role in mechanically induced vascular remodeling of the arterial tree in skeletal muscle.

We and others have shown intermediate‐conductance, Ca 2+ ‐activated K + channel (K Ca 3.1) activation contributes to vascular smooth muscle cell (VSMC) migration. VSMC migration necessitates dynamic changes in integrin‐ECM interaction, including focal adhesion turnover, though little is known regarding the mechanical properties of the integrin/ECM interaction in VSMC and how it is regulated when cells are stimulated with a pro‐migratory agonist. We tested the hypothesis that K Ca 3.1 activation alters matrix‐integrin adhesion. We used atomic force microscopy (AFM) to determine the binding properties of fibronectin (FN)/α5β1‐integrin and collagen I/ α 1 β 1 ‐integrin in mouse aortic VSMC (MAVSMC). Sub‐confluent MAVSCM were serum‐starved overnight and treated with PDGF‐BB (10 ng/ml) with and without the K Ca 3.1 inhibitor, TRAM‐34 (100 nM). AFM probes with a FN‐ or collagen I‐coated beads were brought into contact with the cell surface for 2 min and the probes subsequently pulled away from the cell surface in the z‐axis. With FN coated‐beads, PDGF‐BB increased total force required to break adhesion of bead with the MAVSMC compared to control (2539 ± 705 vs. 1093 ± 114 pN) indicative of increased adhesion to FN. This increase was inhibited by TRAM‐34 and reduced to control levels (1068 ± 182 pN). With collagen I coated‐beads, PDGF‐BB had no effect on the force required to break adhesion with the MAVSMC compared to control (1036 ± 263 vs. 1207 ± 237 pN), however TRAM‐34 did reduce adhesion to collagen I compared to control (636 ± 142 pN). Thus, K Ca 3.1 activation stimulates acute integrin/ECM interaction in an integrin and ECM specific manner in response to PDGF‐BB.

Accelerated development of coronary atherosclerosis is a defining characteristic of familial hypercholesterolemia (FH). However, the recent data highlight a significant cardiovascular risk prior to the development of critical coronary stenosis. We, therefore, examined the hypothesis that FH produces coronary microvascular dysfunction and impairs coronary vascular control at rest and during exercise in a swine model of FH. Coronary vascular responses to drug infusions and exercise were examined in chronically instrumented control and FH swine. FH swine exhibited ~tenfold elevation of plasma cholesterol and diffuse coronary atherosclerosis (20–60 % plaque burden). Similar to our recent findings in the systemic vasculature in FH swine, coronary smooth muscle nitric oxide sensitivity was increased in vivo and in vitro with maintained endothelium-dependent vasodilation in vivo in FH. At rest and during exercise, FH swine exhibited increased myocardial O2 extraction resulting in reduced coronary venous SO2 and PO2 versus control. During exercise in FH swine, the transmural distribution of coronary blood flow was unchanged; however, a shift toward anaerobic cardiac metabolism was revealed by increased coronary arteriovenous H+ concentration gradient. This shift was associated with a worsening of cardiac efficiency (relationship between cardiac work and O2 consumption) in FH during exercise owing, in part, to a generalized reduction in stroke volume which was associated with increased left atrial pressure in FH. Our data highlight a critical role for coronary microvascular dysfunction as a contributor to impaired myocardial O2 balance, cardiac ischemia, and impaired cardiac function prior to the development of critical coronary stenosis in FH.

Hormone replacement therapy is known to have significant effects on protein expression and function in brain regions important for blood pressure regulation. The nucleus of the solitary tract (NTS) is a key brainstem region involved blood pressure control and has been found to have sex differences in microglia activation during hypertension. The goal of the present study was to identify genes in the NTS sensitive to estradiol (E2) hormone replacement and may be involved in a hypertension induced brain inflammatory response. Affymetrix microarrays were used to compare mRNA levels in the NTS in ovariectomized female rats receiving either E2 (n=12) or saline (n=12) administered for 21 days via subcutaneous pellet implant. Using a combination of criteria, we identified a small set of genes on the array (6 of 431 significantly changed genes) with the largest significant responses to E2 and also known to be related to inflammation. Of these 6 genes, 5 were down-regulated by E2 with negative fold changes including: -IL-1 type 1 receptor, -47.2 fold change; LPS binding protein, -10.2 fold change; T-cell receptor B-chain, -6.4 fold change; tenascin-C, -2.4 fold change and CD5 lymphocyte antigen, -1.97 fold change. The one significant fold increase was for heat-shock protein (hsp) 70, + 3.1 fold change. These results may suggest that E2 hormone replacement therapy may affect brain blood pressure regulation by down-regulating pro-inflammatory genes and up-regulating protective genes, such as hsp 70. NIH HL6226, HL098207.