Zachary P. Neeb

Epicardial Perivascular Adipose-Derived Leptin Exacerbates Coronary Endothelial Dysfunction in Metabolic Syndrome via a Protein Kinase C-β Pathway

Objective—Factors released by perivascular adipose tissue (PVAT) disrupt coronary endothelial function via phosphorylation of endothelial NO synthase by protein kinase C (PKC)-&bgr;. However, our understanding of how PVAT potentially contributes to coronary disease as a complication of obesity/metabolic syndrome (MetS) remains limited. The current study investigated whether PVAT-derived leptin impairs coronary vascular function via PKC-&bgr; in MetS. Methods and Results—Coronary arteries with and without PVAT were collected from lean or MetS Ossabaw miniature swine for isometric tension studies. Endothelial-dependent vasodilation to bradykinin was significantly reduced in MetS. PVAT did not affect bradykinin-mediated dilation in arteries from lean swine but significantly exacerbated endothelial dysfunction in arteries from MetS swine. PVAT-induced impairment was reversed by inhibition of either PKC-&bgr; with ruboxistaurin (Eli Lilly and Company, Indianapolis, Ind) or leptin receptor signaling with a recombinant, pegylated leptin antagonist. Western blot and immunohistochemical analyses demonstrated increased PVAT-derived leptin and coronary leptin receptor density with MetS. Coronary PKC-&bgr; activity was increased in both MetS arteries exposed to PVAT and lean arteries exposed to leptin. Finally, leptin-induced endothelial dysfunction was reversed by ruboxistaurin. Conclusion—Increases in epicardial PVAT leptin exacerbate coronary endothelial dysfunction in MetS via a PKC-&bgr;-dependent pathway. These findings implicate PVAT-derived leptin as a potential contributor to coronary atherogenesis in MetS.

Impaired capsaicin-induced relaxation of coronary arteries in a porcine model of the metabolic syndrome

Recent studies implicate channels of the transient receptor potential vanilloid family (e.g., TRPV1) in regulating vascular tone; however, little is known about these channels in the coronary circulation. Furthermore, it is unclear whether metabolic syndrome alters the function and/or expression of TRPV1. We tested the hypothesis that TRPV1 mediates coronary vasodilation through endothelium-dependent mechanisms that are impaired by the metabolic syndrome. Studies were conducted on coronary arteries from lean and obese male Ossabaw miniature swine. In lean pigs, capsaicin, a TRPV1 agonist, relaxed arteries in a dose-dependent manner (EC50 = 116 +/- 41 nM). Capsaicin-induced relaxation was blocked by the TRPV1 antagonist capsazepine, endothelial denudation, inhibition of nitric oxide synthase, and K+ channel antagonists. Capsaicin-induced relaxation was impaired in rings from pigs with metabolic syndrome (91 +/- 4% vs. 51 +/- 10% relaxation at 100 microM). TRPV1 immunoreactivity was prominent in coronary endothelial cells. TRPV1 protein expression was decreased 40 +/- 11% in obese pigs. Capsaicin (100 microM) elicited divalent cation influx that was abolished in endothelial cells from obese pigs. These data indicate that TRPV1 channels are functionally expressed in the coronary circulation and mediate endothelium-dependent vasodilation through a mechanism involving nitric oxide and K+ channels. Impaired capsaicin-induced vasodilation in the metabolic syndrome is associated with decreased expression of TRPV1 and cation influx.

TMEM16A/ANO1 Channels Contribute to the Myogenic Response in Cerebral Arteries

Rationale: Pressure-induced arterial depolarization and constriction (the myogenic response) is a smooth muscle cell (myocyte)-specific mechanism that controls regional organ blood flow and systemic blood pressure. Several different nonselective cation channels contribute to pressure-induced depolarization, but signaling mechanisms involved are unclear. Similarly uncertain is the contribution of anion channels to the myogenic response and physiological functions and mechanisms of regulation of recently discovered transmembrane 16A (TMEM16A), also termed Anoctamin 1, chloride (Cl−) channels in arterial myocytes. Objective: To investigate the hypothesis that myocyte TMEM16A channels control membrane potential and contractility and contribute to the myogenic response in cerebral arteries. Methods and Results: Cell swelling induced by hyposmotic bath solution stimulated Cl− currents in arterial myocytes that were blocked by TMEM16A channel inhibitory antibodies, RNAi-mediated selective TMEM16A channel knockdown, removal of extracellular calcium (Ca2+), replacement of intracellular EGTA with BAPTA, a fast Ca2+ chelator, and Gd3+ and SKF-96365, nonselective cation channel blockers. In contrast, nimodipine, a voltage-dependent Ca2+ channel inhibitor, or thapsigargin, which depletes intracellular Ca2+ stores, did not alter swelling-activated TMEM16A currents. Pressure-induced (−40 mm Hg) membrane stretch activated ion channels in arterial myocyte cell–attached patches that were inhibited by TMEM16A antibodies and were of similar amplitude to recombinant TMEM16A channels. TMEM16A knockdown reduced intravascular pressure-induced depolarization and vasoconstriction but did not alter depolarization-induced (60 mmol/L K+) vasoconstriction. Conclusions: Membrane stretch activates arterial myocyte TMEM16A channels, leading to membrane depolarization and vasoconstriction. Data also provide a mechanism by which a local Ca2+ signal generated by nonselective cation channels stimulates TMEM16A channels to induce myogenic constriction.

Exercise training decreases store-operated Ca2+entry associated with metabolic syndrome and coronary atherosclerosis

AIMS Stenting attenuates restenosis, but accelerated coronary artery disease (CAD) adjacent to the stent (peri-stent CAD) remains a concern in metabolic syndrome (MetS). Smooth muscle cell proliferation, a major mechanism of CAD, is mediated partly by myoplasmic Ca2+ dysregulation and store-operated Ca2+ entry (SOCE) via canonical transient receptor potential 1 (TRPC1) channels is proposed to play a key role. Exercise is known to prevent Ca2+ dysregulation in CAD. We tested the hypothesis that MetS increases SOCE and peri-stent CAD and exercise attenuates these events. METHODS AND RESULTS Groups (n = 9 pigs each) were (i) healthy lean Ossabaw swine fed standard chow, (ii) excess calorie atherogenic diet fed (MetS), and (iii) aerobically exercise trained starting after 50 weeks of development of MetS (XMetS). Bare metal stents were placed after 54 weeks on diets, and CAD and SOCE were assessed 4 weeks later. Coronary cells were dispersed proximal to the stent (peri-stent) and from non-stent segments, and fura-2 fluorescence was used to assess SOCE, which was verified by Ni2+ blockade and insensitivity to nifedipine. XMetS pigs had increased physical work capacity and decreased LDL/HDL (P < 0.05), but no attenuation of robust insulin resistance, glucose intolerance, hypertriglyceridaemia, or hypertension. CAD was greater in peri-stented vs. non-stented artery segments. MetS had the greatest CAD, SOCE, and TRPC1 and STIM1 mRNA and protein expression, which were all attenuated in XMetS. CONCLUSION This is the first report of the protective effect of exercise on native CAD, peri-stent CAD, SOCE, and molecular expression of TRPC1, STIM1, and Orai1 in MetS.

Impaired function of coronary BKCa channels in metabolic syndrome

The role of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in regulation of coronary microvascular function is widely appreciated, but molecular and functional changes underlying the deleterious influence of metabolic syndrome (MetS) have not been determined. Male Ossabaw miniature swine consumed for 3-6 mo a normal diet (11% kcal from fat) or an excess-calorie atherogenic diet that induces MetS (45% kcal from fat, 2% cholesterol, 20% kcal from fructose). MetS significantly impaired coronary vasodilation to the BK(Ca) opener NS-1619 in vivo (30-100 microg) and reduced the contribution of these channels to adenosine-induced microvascular vasodilation in vitro (1-100 microM). MetS reduced whole cell penitrem A (1 microM)-sensitive K(+) current and NS-1619-activated (10 microM) current in isolated coronary vascular smooth muscle cells. MetS increased the concentration of free intracellular Ca(2+) and augmented coronary vasoconstriction to the L-type Ca(2+) channel agonist BAY K 8644 (10 pM-10 nM). BK(Ca) channel alpha and beta(1) protein expression was increased in coronary arteries from MetS swine. Coronary vascular dysfunction in MetS is related to impaired BK(Ca) channel function and is accompanied by significant increases in L-type Ca(2+) channel-mediated coronary vasoconstriction.

TMEM16A channels generate Ca2+-activated Cl− currents in cerebral artery smooth muscle cells

Transmembrane protein (TMEM)16A channels are recently discovered membrane proteins that display electrophysiological properties similar to classic Ca(2+)-activated Cl(-) (Cl(Ca)) channels in native cells. The molecular identity of proteins that generate Cl(Ca) currents in smooth muscle cells (SMCs) of resistance-size arteries is unclear. Similarly, whether cerebral artery SMCs generate Cl(Ca) currents is controversial. Here, using molecular biology and patch-clamp electrophysiology, we examined TMEM16A channel expression and characterized Cl(-) currents in arterial SMCs of resistance-size rat cerebral arteries. RT-PCR amplified transcripts for TMEM16A but not TMEM16B-TMEM16H, TMEM16J, or TMEM16K family members in isolated pure cerebral artery SMCs. Western blot analysis using an antibody that recognized recombinant (r)TMEM16A channels detected TMEM16A protein in cerebral artery lysates. Arterial surface biotinylation and immunofluorescence indicated that TMEM16A channels are located primarily within the arterial SMC plasma membrane. Whole cell Cl(Ca) currents in arterial SMCs displayed properties similar to those generated by rTMEM16A channels, including Ca(2+) dependence, current-voltage relationship linearization by an elevation in intracellular Ca(2+) concentration, a Nerstian shift in reversal potential induced by reducing the extracellular Cl(-) concentration, and a negative reversal potential shift when substituting extracellular I(-) for Cl(-). A pore-targeting TMEM16A antibody similarly inhibited both arterial SMC Cl(Ca) and rTMEM16A currents. TMEM16A knockdown using small interfering RNA also inhibited arterial SMC Cl(Ca) currents. In summary, these data indicate that TMEM16A channels are expressed, insert into the plasma membrane, and generate Cl(Ca) currents in cerebral artery SMCs.

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells.

Metabolic syndrome reduces the contribution of K+ channels to ischemic coronary vasodilation

This investigation tested the hypothesis that metabolic syndrome decreases the relative contribution of specific K(+) channels to coronary reactive hyperemia. Ca(2+)-activated (BK(Ca)), voltage-activated (K(V)), and ATP-dependent (K(ATP)) K(+) channels were investigated. Studies were conducted in anesthetized miniature Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) for 20 wk. The latter diet induces metabolic syndrome, increasing body weight, fasting glucose, total cholesterol, and triglyceride levels. Ischemic vasodilation was determined by the coronary flow response to a 15-s occlusion before and after cumulative administration of antagonists for BK(Ca) (penitrem A; 10 microg/kg iv), K(V) (4-aminopyridine; 0.3 mg/kg iv) and K(ATP) (glibenclamide; 1 mg/kg iv) channels. Coronary reactive hyperemia was diminished by metabolic syndrome as the repayment of flow debt was reduced approximately 30% compared with lean swine. Inhibition of BK(Ca) channels had no effect on reactive hyperemia in either lean or metabolic syndrome swine. Subsequent inhibition of K(V) channels significantly reduced the repayment of flow debt ( approximately 25%) in both lean and metabolic syndrome swine. Additional blockade of K(ATP) channels further diminished ( approximately 45%) the repayment of flow debt in lean but not metabolic syndrome swine. These data indicate that the metabolic syndrome impairs coronary vasodilation in response to cardiac ischemia via reductions in the contribution of K(+) channels to reactive hyperemia.

Dynamic micro- and macrovascular remodeling in coronary circulation of obese Ossabaw pigs with metabolic syndrome

Previous studies from our laboratory showed that coronary arterioles from type 2 diabetic mice undergo inward hypertrophic remodeling and reduced stiffness. The aim of the current study was to determine if coronary resistance microvessels (CRMs) in Ossabaw swine with metabolic syndrome (MetS) undergo remodeling distinct from coronary conduit arteries. Male Ossabaw swine were fed normal (n = 7, Lean) or hypercaloric high-fat (n = 7, MetS) diets for 6 mo, and then CRMs were isolated and mounted on a pressure myograph. CRMs isolated from MetS swine exhibited decreased luminal diameters (126 ± 5 and 105 ± 9 μm in Lean and MetS, respectively, P < 0.05) with thicker walls (18 ± 3 and 31 ± 3 μm in Lean and MetS, respectively, P < 0.05), which doubled the wall-to-lumen ratio (14 ± 2 and 30 ± 2 in Lean and MetS, respectively, P < 0.01). Incremental modulus of elasticity (IME) and beta stiffness index (BSI) were reduced in CRMs isolated from MetS pigs (IME: 3.6 × 10(6) ± 0.7 × 10(6) and 1.1 × 10(6) ± 0.2 × 10(6) dyn/cm(2) in Lean and MetS, respectively, P < 0.001; BSI: 10.3 ± 0.4 and 7.3 ± 1.8 in Lean and MetS, respectively, P < 0.001). BSI in the left anterior descending coronary artery was augmented in pigs with MetS. Structural changes were associated with capillary rarefaction, decreased hyperemic-to-basal coronary flow velocity ratio, and augmented myogenic tone. MetS CRMs showed a reduced collagen-to-elastin ratio, while immunostaining for the receptor for advanced glycation end products was selectively increased in the left anterior descending coronary artery. These data suggest that MetS causes hypertrophic inward remodeling of CRMs and capillary rarefaction, which contribute to decreased coronary flow and myocardial ischemia. Moreover, our data demonstrate novel differential remodeling between coronary micro- and macrovessels in a clinically relevant model of MetS.

Contribution of BKCa channels to local metabolic coronary vasodilation: effects of metabolic syndrome

This investigation was designed to examine the hypothesis that impaired function of coronary microvascular large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in metabolic syndrome (MetS) significantly attenuates the balance between myocardial oxygen delivery and metabolism at rest and during exercise-induced increases in myocardial oxygen consumption (MVo(2)). Studies were conducted in conscious, chronically instrumented Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) that induces many common features of MetS. Data were collected under baseline/resting conditions and during graded treadmill exercise before and after selective blockade of BK(Ca) channels with penitrem A (10 microg/kg iv). We found that the exercise-induced increases in blood pressure were significantly elevated in MetS swine. No differences in baseline cardiac function or heart rate were noted. Induction of MetS produced a parallel downward shift in the relationship between coronary venous Po(2) and MVo(2) (P < 0.001) that was accompanied by a marked release of lactate (negative lactate uptake) as MVo(2) was increased with exercise (P < 0.005). Inhibition of BK(Ca) channels with penitrem A did not significantly affect blood pressure, heart rate, or the relationship between coronary venous Po(2) and MVo(2) in lean or MetS swine. These data indicate that BK(Ca) channels are not required for local metabolic control of coronary blood flow under physiological (lean) or pathophysiological (MetS) conditions. Therefore, diminished function of BK(Ca) channels does not contribute to the impairment of myocardial oxygen-supply demand balance in MetS.