Loss of fatty acid binding protein 3 ameliorates lipopolysaccharide-induced inflammation and endothelial dysfunction.

Circulating fatty acid-binding protein 3 (FABP3) is an effective biomarker of myocardial injury and peripheral artery disease (PAD). The endothelium, which forms the inner most layer of every blood vessel, is exposed to higher levels of FABP3 in PAD or following myocardial injury, but the pathophysiological role of endothelial FABP3, the effect of FABP3 exposure on endothelial cells, and related mechanisms are unknown. Here, we aimed to evaluate the pathophysiological role of endothelial FABP3 and related mechanisms in vitro. Our molecular and functional in vitro analyses show that (1) FABP3 is basally expressed in endothelial cells; (2) inflammatory stress in the form of lipopolysaccharide (LPS) upregulated endothelial FABP3 expression; (3) loss of endogenous FABP3 protected endothelial cells against LPS-induced endothelial dysfunction; however, exogenous FABP3 exposure exacerbated LPS-induced inflammation; (4) loss of endogenous FABP3 protected against LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling pathways. Together, these findings suggest that gain-of endothelial FABP3 exacerbates, whereas loss-of endothelial FABP3 inhibits LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling. We propose that an increased circulating FABP3 in myocardial injury or PAD patients may be detrimental to endothelial function, and therefore, therapies aimed at inhibiting FABP3 may improve endothelial function in diseased states.

Endothelium dysfunction in hind limb arteries of male Zucker Diabetic-Sprague Dawley rats.

Endothelium dysfunction produces peripheral vascular disease comorbidities in type 2 diabetes, including hypertension, and critical limb ischemia. In this study we aimed to test endothelial dysfunction, the vasodilator effects of a proteinase-activated receptor 2 (PAR2) agonist (2fLIGRLO), and thromboxane A2 synthase inhibitor (ozagrel) on PAR2 vasodilation in hind limb arteries ex vivo, using Zucker Diabetic-Sprague Dawley (ZDSD) rats, a model of type 2 diabetes. Male Sprague Dawley rats (SD) and ZDSD were fed a high-fat content 'Western diet' from 16 to 20 weeks of age (wks) then fed a standard laboratory diet. We identified diabetic ZDSD rats by two consecutive blood glucose measurements > 12.5 mM, based on weekly monitoring. We used acetylcholine, 2fLIGRLO, and nitroprusside with wire-myograph methods to compare relaxations of femoral, and saphenous arteries from diabetic ZDSD (21-23 wks) to age-matched normoglycemic SD. All arteries showed evidence of endothelium dysfunction using acetylcholine (reduced maximum relaxations, reduced sensitivity), and higher sensitivities to 2fLIGRLO, and nitroprusside in ZDSD vs SD. Ozagrel treatment of ZDSD distal segments, and end-branches of saphenous arteries decreased their sensitivities to 2fLIGRLO. We tested aortas for altered expression of endothelium-specific gene targets using PCR array and qPCR. PAR2, and placental growth factor gene transcripts were 1.5, and 4-times higher in ZDSD than SD aortas. Hind limb arteries of ZDSD exhibit endothelium dysfunction having less GPCR agonist induced vasodilation by endothelial NO-release. Different expression of several endothelial genes in ZDSD vs SD aortas, including PAR2, suggests altered inflammatory, and angiogenesis signaling pathways in the endothelium of ZDSD.

Characterization of Endothelium-Dependent Relaxation in the Saphenous Artery and Its Caudal Branches in Young and Old Adult Sprague Dawley Rats.

Ageing is associated with reduced endothelium-derived nitric oxide (NO) production in the femoral artery of Sprague Dawley (SD) rats. In the current study, we examined endothelium-dependent relaxation (EDR) in the saphenous artery and its caudal branches. We used acetylcholine and the Proteinase-Activated receptor-2 (PAR2)-specific agonist (2fLIGRLO) with nitroarginine methylester (L-NAME) to assess EDR in two groups of male SD rats (age in weeks: young, 10-12; old, 27-29). Acetylcholine and 2fLIGRLO were potent NO-dependent relaxant agents in all arteries. For all arteries, EDR by acetylcholine decreased significantly in old compared to young SD rats. Interestingly, PAR2-induced EDR of proximal saphenous artery segments and caudal branches decreased significantly in old compared to young, but did not differ for the in-between middle and distal ends of the saphenous artery. L-NAME treatment increased subsequent contractions of proximal and middle segments of saphenous arteries by phenylephrine and U46619 in young, but not in old, SD rats. We conclude the SD saphenous artery and caudal branches exhibit regional characteristics that differ in response to specific EDR agonists, endothelial NO synthase inhibitor, and changes to endothelium function with increased age, which are, in part, attributed to decreased sensitivity of vascular smooth muscle to the gaseous transmitter NO.

Modulation of Vasomotor Function by Perivascular Adipose Tissue of Renal Artery Depends on Severity of Arterial Dysfunction to Nitric Oxide and Severity of Metabolic Parameters.

Perivascular adipose tissue (PVAT) enhances vascular relaxation of mesenteric arteries in SHRSP.Z-Leprfa/IzmDmcr rats (SPZF), a metabolic syndrome model. We investigated and compared the effects of PVAT on the renal artery in SPZF with those on SHR/NDmcr-cp rats (CP). Renal arteries with and without PVAT were isolated from 23-week-old SPZF and CP. The effects of PVAT on acetylcholine- and nitroprusside-induced relaxation were examined using bioassays with phenylephrine-contracted arterial rings. Acetylcholine-induced relaxations without PVAT in SPZF and CP were 0.7- and 0.5-times lower in females than in males, respectively. In the presence of PVAT, acetylcholine-induced relaxations increased 1.4- and 2-times in male and female CP, respectively, but did not differ in SPZF. Nitroprusside-induced relaxation with and without PVAT was 0.7-times lower in female than in male SPZF but did not differ in CP. Angiotensin-II type-1 receptor (AT1R)/AT1R-associated protein mRNA ratios were lower in CP than in the SPZF and negatively correlated with the difference in arterial relaxation with and without PVAT. The effects of renal artery PVAT differed between the SPZF and CP groups. Higher levels of enhanced AT1R activity in SPZF PVAT may drive these differences by impairing the vascular smooth muscle responses to nitric oxide.

Perivascular Adipose Tissue Compensation for Endothelial Dysfunction in the Superior Mesenteric Artery of Female SHRSP.Z-Leprfa/IzmDmcr Rats.

Regulation of arterial tone by perivascular adipose tissue (PVAT) differs between sexes. In male SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF), PVAT exerts a compensatory relaxation effect for the loss of endothelium-mediated vasorelaxation, which occurs during the early stages of metabolic syndrome. However, this effect deteriorates by 23 weeks of age. Here, therefore, we compared the effects of PVAT in female and male SHRSP.ZF. Acetylcholine-induced relaxation in superior mesenteric artery without PVAT did not differ between 23-week-old females and males. However, the presence of PVAT enhanced relaxation in 23-week-old females, but not in males. The mRNA levels of angiotensin II type 1 receptor (AT1R) in PVAT did not differ between sexes, but AT1R-associated protein (ATRAP) and apelin levels were higher in females than in males. We observed a positive relationship between differences in artery relaxation with and without PVAT and ATRAP or apelin mRNA levels. In 30-week-old females, PVAT-enhanced relaxation disappeared, and mRNA levels of AT1R increased, while apelin levels decreased compared to 23-week-old females. These results demonstrated that in SHRSP.ZF, PVAT compensation for endothelium dysfunction extended to older ages in females than in males. Apelin and AT1R/ATRAP expression in PVAT may be predictors of favorable effects.

Zucker Diabetic-Sprague Dawley (ZDSD) rat: Type 2 diabetes translational research model.

NEW FINDINGS: What is the topic of this review? The Zucker Diabetic-Sprague Dawley (ZDSD) rat is in the early adoption phase of use by researchers in the fields of diabetes, including prediabetes, obesity and metabolic syndrome. It is essential that physiology researchers choose preclinical models that model human type 2 diabetes appropriately and are aware of the limitations on experimental design. What advances does it highlight? Our review of the scientific literature finds that although sex, age and diets contribute to variability, the ZDSD phenotype and disease progression model the characteristics of humans who have prediabetes and diabetes, including co-morbidities. ABSTRACT: Type 2 diabetes (T2D) is a prevalent disease and a significant concern for global population health. For persons with T2D, clinical treatments target not only the characteristics of hyperglycaemia and insulin resistance, but also co-morbidities, such as obesity, cardiovascular and renal disease, neuropathies and skeletal bone conditions. The Zucker Diabetic-Sprague Dawley (ZDSD) rat is a rodent model developed for experimental studies of T2D. We reviewed the scientific literature to highlight the characteristics of T2D development and the associated phenotypes, such as metabolic syndrome, cardiovascular complications and bone and skeletal pathologies in ZDSD rats. We found that ZDSD phenotype characteristics are independent of leptin receptor signalling. The ZDSD rat develops prediabetes, then progresses to overt diabetes that is accelerated by introduction of a timed high-fat diet. In male ZDSD rats, glycated haemoglobin (HbA1c) increases at a constant rate from 7 to >30 weeks of age. Diabetic ZDSD rats are moderately hypertensive compared with other rat strains. Diabetes in ZDSD rats leads to endothelial dysfunction in specific vasculatures, impaired wound healing, decreased systolic and diastolic cardiac function, neuropathy and nephropathy. Changes to bone composition and the skeleton increase the risk of bone fractures. Zucker Diabetic-Sprague Dawley rats have not yet achieved widespread use by researchers. We highlight sex-related differences in the ZDSD phenotype and gaps in knowledge for future studies. Overall, scientific data support the premise that the phenotype and disease progression in ZDSD rats models the characteristics in humans. We conclude that ZDSD rats are an advantageous model to advance understanding and discovery of treatments for T2D through preclinical research.

Can Myogenic Tone Protect Endothelial Function? Integrating Myogenic Activation and Dilator Reactivity for Cerebral Resistance Arteries in Metabolic Disease.

The obese Zucker rat (OZR) manifests multiple risk factors for impaired cerebrovascular function, including hypertension and insulin resistance although how they combine to produce integrated vascular function is unclear. As studies have suggested that myogenic activation (MA) severity for middle cerebral arteries (MCAs) may be proportional to hypertension severity, we hypothesized that MA will negatively correlate with dilator reactivity in OZR. MA of MCA from OZR was divided into low, medium, and high based on the slope of MA, while MCA reactivity and vascular metabolite bioavailability were assessed in all groups. Endothelium-dependent dilation of MCA in OZR was attenuated and correlated with the MA slope. Treatment of OZR MCA with TEMPOL (antioxidant) improved dilation in low or medium MA groups, but had less impact on high MA. Alternatively, treatment with gadolinium to normalize MA in OZR had reduced impact on dilator reactivity in MCA from low and medium MA groups, but improved responses in the high group. Treatment with both agents resulted in dilator responses that were comparable across all groups. These results suggest that, under conditions with stronger MA, endothelial function may receive some protection despite the environment, potentially from the ability of MCA to reduce wall tension despite increased pressure.

A Sodium Glucose Cotransporter 2 Inhibitor Fails to Improve Perivascular Adipose Tissue-Mediated Modulation of Vasodilation and Cardiac Function in Rats With Metabolic Syndrome.

Arterial perivascular adipose tissue (PVAT) can elicit vasodilator signals complementary to those elicited by the endothelium in SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats, an animal model of metabolic syndrome (MetS). Here, we tested whether a glucose cotransporter 2 inhibitor (SGLT2-i; tofogliflozin) increased this PVAT effect to prevent the deterioration of cardiac function in aging SHRSP.ZF rats. Tofogliflozin treatments (1 or 10 mg/kg/day) or vehicle (control) were administered for 10 weeks by oral gavage to SHRSP.ZF rats, starting at 13 weeks of age. At 23 weeks of age, glucose levels in the serum and urine (24 h after the last administration) were determined using commercial kits. Vasodilator responsiveness of PVAT-surrounded or PVAT-free superior mesenteric arteries was determined using acetylcholine with organ-bath methods. Cardiac ventricular function and coronary flow were determined using Langendorff heart preparations. Serum and urine glucose levels in SGLT2-i treatment groups did not differ from those in the controls, but the ratios of glycated to non-glycated albumin were lower than those in the controls. Tofogliflozin treatments did not alter relaxations in the presence of PVAT or affect relaxations of PVAT-free arteries. Left ventricular systolic pressures, maximum rate of pressure decline, and coronary flow in ex vivo hearts did not differ among the treatment groups. PVAT effects and cardiac dysfunction were not altered by tofogliflozin treatment in SHRSP.ZF rats with MetS. These results do not provide strong evidence to support the use of SGLT2-i as a cardiovascular protective therapy in MetS, which occurs prior to the onset of type 2 diabetes.

Activation of protease-activated receptor 2 is associated with blood pressure regulation and proteinuria reduction in metabolic syndrome.

Metabolic syndrome (MetS) increases the risk of kidney disease. In SHRSP.Z-Leprfa /IzmDmcr (SHRSP.ZF) rats with MetS, protease-activated receptor 2 (PAR2)-mediated vasorelaxation is preserved in the aorta at 20 weeks of age (weeks) via enhancement of nitric oxide production but impaired at 30 weeks by oxidative stress. However, impairment of PAR2-mediated vasorelaxation of renal arteries and its possible implications for kidney disease are unclear. We used organ baths to assess PAR2-mediated vasorelaxation of isolated renal arteries, colorimetric methods to measure urinary protein levels as an index of renal function, and western blot to determine expression of PAR2 and nephrin proteins in the kidneys of SHRSP.ZF rats at 10, 20, and 30 weeks. We assessed renal arteries and kidney function for effects of orally administered GB88, a pathway-dependent PAR2 antagonist, from 10 to 18 weeks, and azilsartan, an angiotensin II type 1 receptor blocker, from 13 to 23 weeks. PAR2-mediated vasorelaxation was slightly lower at 20 weeks and attenuated significantly at 30 weeks compared with those at 10 weeks. Urinary protein levels were increased at 20 and 30 weeks. Decreased protein expression of PAR2 and nephrin in the kidney were observed at 30 weeks. Administration of GB88 increased blood pressure (BP) and proteinuria. Azilsartan reduced the high BP and the impaired PAR2-mediated vasorelaxation, but did not restore the increase in urinary protein levels and decreased PAR2 and nephrin protein expression in the kidney. PAR2 activation in the kidney may be associated with maintenance of BP and urinary protein excretion in MetS.

Angiotensin II Type 1 Receptor Antagonist Azilsartan Restores Vascular Reactivity Through a Perivascular Adipose Tissue-Independent Mechanism in Rats with Metabolic Syndrome.

PURPOSE: Perivascular adipose tissues (PVAT) are involved in the regulation of vascular tone. In mesenteric arteries, the compensatory vasodilatory effects of PVAT appear when vascular relaxation is impaired and disappear at around 23 weeks of age in SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome (MetS). The renin-angiotensin system is involved in the development of endothelium and vascular dysfunction. Therefore, we investigated whether azilsartan, a potent angiotensin II type 1 (AT1) receptor antagonist, can protect against the deterioration of the PVAT compensatory vasodilator function that occurs with aging in MetS. METHODS: Two age groups of SHRSP.ZF rats (13 and 20 weeks of age) were administered azilsartan or vehicle through oral gavage once daily for 10 weeks. The vasodilation response of the isolated superior-mesenteric arteries upon addition of endothelium-dependent and -independent agonists was determined in the presence or absence of PVAT using organ bath methods. RESULTS: In vivo treatment with azilsartan improved the acetylcholine-induced vasodilation in mesenteric arteries with and without PVAT at both time-points. The mRNA levels of AT1 receptor and AT1 receptor-associated protein were unchanged in PVAT upon azilsartan treatment. Furthermore, in vitro treatment with azilsartan (0.1 and 0.3 µM for 30 min) did not affect the compensatory effect of PVAT on vasodilation in response to acetylcholine in SHRSP.ZF rat mesenteric arteries. CONCLUSIONS: Our results provide evidence supporting the use of azilsartan for the long-term protection against vascular dysfunctions in MetS. Azilsartan did not improve the dysfunction of PVAT-mediated modulation of vascular tone during MetS. The protective effect of azilsartan is mediated by restoring the endothelium- and vascular smooth muscle-mediated mechanisms.

Endothelium-dependent impairments to cerebral vascular reactivity with type 2 diabetes mellitus in the Goto-Kakizaki rat.

Type 2 diabetes mellitus (T2DM) is a prevalent pathology associated with elevated cerebrovascular disease risk. We determined wall mechanics and vascular reactivity in ex vivo middle cerebral arteries (MCA) from male Goto-Kakizaki rats (GK; ~17 wk old) versus control Wistar Kyoto rats (WKY) to test the hypothesis that the diabetic environment in GK, in the absence of obesity and other comorbidities, leads to endothelial dysfunction and impaired vascular tone regulation. Dilation of MCA following challenge with acetylcholine and hypoxia was blunted in MCA from GK versus WKY, due to lower nitric oxide bioavailability and altered arachidonic acid metabolism, whereas myogenic activation and constrictor responses to serotonin were unchanged. MCA wall distensibility and cross-sectional area were not different between GK and WKY, suggesting that wall mechanics were unchanged at this age, supported by the determination that MCA dilation to sodium nitroprusside was also intact. With the use of ex vivo aortic rings as a bioassay, altered vascular reactivity determined in MCA was paralleled by relaxation responses in artery segments from GK, whereas measurements of vasoactive metabolite production indicated a loss of nitric oxide and prostacyclin bioavailability and an increased thromboxane A2 production with both methacholine challenge and hypoxia. These results suggest that endothelium-dependent dilator reactivity of MCA in GK is impaired with T2DM, and that this impairment is associated with the genesis of a prooxidant/pro-inflammatory condition with diabetes mellitus. The restriction of vascular impairments to endothelial function only, at this age and development, provide insight into the severity of multimorbid conditions of which T2DM is only one constituent.

Perivascular Adipose Tissue-Enhanced Vasodilation in Metabolic Syndrome Rats by Apelin and N-Acetyl⁻l-Cysteine-Sensitive Factor(s).

Perivascular adipose tissue (PVAT) can regulate vascular tone. In mesenteric arteries of SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF) with metabolic syndrome, vascular dysfunction is compensated by PVAT-dependent mechanisms that disappear with increasing age. In this study, we investigated the mechanisms of the age-related changes and responsible factor(s) involved in the enhancing effects of mesenteric arterial PVAT in SHRSP.ZF. Acetylcholine- and sodium nitroprusside-induced relaxations of isolated arteries were greater with PVAT than without PVAT at 17 and 20 weeks of age (wks), and as expected, this enhancement by the presence of PVAT disappeared at 23 wks. PVAT mRNA levels of angiotensin II type 1 (AT1) receptor-associated protein was less and AT1 receptor was unchanged at 23 wks when compared to 20 wks. At 20 wks, the enhanced acetylcholine-induced relaxation by the presence of PVAT was inhibited by N-acetyl-l-cysteine (NAC). Acetylcholine-induced relaxation of arteries without PVAT was increased in the presence of exogenously added apelin. PVAT mRNA level of apelin was higher in SHRSP.ZF than in control Wistar-Kyoto rats, and the level was decreased with aging. These results suggest that AT1 receptor activation in PVAT, and changes in the regulation of apelin and a NAC-sensitive factor are related to the age-dependent deterioration of the vasodilation enhancing effects of mesenteric arterial PVAT in SHRSP.ZF.

Progression of Time-Dependent Changes to the Mechanisms of Vasodilation by Protease-Activated Receptor 2 in Metabolic Syndrome.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteases released from tissues or by synthetic peptide ligands administered pharmacologically. Its wide expression in the cardiovascular system, particularly within the endothelium, vasodilation activity, and link to increased expression of inflammatory cytokines positions PAR2 as a potentially important regulator of vascular pathology under conditions of tissue inflammation, and injury; and thus, a pharmaceutical target for new therapeutics. Obesity is considered a chronic low-grade systemic inflammatory condition as inflammatory cytokines released from adipocytes are closely related to development of metabolic syndrome and related disorders. Our work over the past five-years has focused on the changes in vasomotor functions of PAR2 in metabolic syndrome, using an animal model known as the SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF). In young SHRSP.ZF that had already developed impaired responses to nitric oxide, we reported that PAR2-induced endothelium-dependent vasodilation is preserved. However, this PAR2 vasodilation decreased with increasing age and further chronic exposure to the conditions of metabolism disorder. These findings raise the possibility that PAR2 regulates tissue perfusion and can protect organs from injury, which is an increasing clinical concern at later stages of metabolic syndrome. Here we present our studies on the time-dependent changes in vasoreactivity to PAR2 in metabolic syndrome and the underlying mechanisms. Furthermore, we discuss the implications of these age-related changes in PAR2 for the cardiovascular system in metabolic syndrome.

Intrauterine growth-restricted Yucatan miniature pigs experience early catch-up growth, leading to greater adiposity and impaired lipid metabolism as young adults.

Early nutrition has critical influences on cardiovascular disease risk in adulthood. The study objectives were to evaluate the impact of low birth weight on fasting and postprandial lipid metabolism and endothelium function in Yucatan miniature pigs. Intrauterine growth-restricted (IUGR) piglets (n = 6; 3 days old, 0.73 ± 0.04 kg) were paired with normal-weight (NW) same-sex littermates (n = 6; 1.11 ± 0.05 kg) and fed milk replacer ad libitum for 4 weeks. Thereafter, all pigs were fed a standard diet ad libitum for 5 h/day with growth, intakes, and blood samples collected for 8 months. At 9 months old, pigs were surgically fitted with venous catheters and an oral fat tolerance test was performed. At 10 months old, pigs were killed and endothelium-dependent and -independent vasodilations of isolated coronary arteries were measured using wire-myographs. IUGR pigs demonstrated catch-up growth (P < 0.05) in body weight and abdominal circumference prior to sexual maturity (<7 months old) and had more (P < 0.05) subcutaneous fat at 10 months old compared with NW pigs. IUGR pigs had consistently higher fasting plasma triglyceride concentrations from 5 to 10 months old and higher liver triglyceride and total cholesterol concentrations at 10 months old (P < 0.05). The fat tolerance test revealed delayed postprandial triglyceride clearance in IUGR pigs, but no differences in plaque formation or vascular reactivity. To conclude, IUGR and early postnatal catch-up growth are associated with increased overall body fat deposition and altered triglyceride metabolism in adult Yucatan miniature swine.

Time-Dependent Differences in the Influence of Perivascular Adipose Tissue on Vasomotor Functions in Metabolic Syndrome.

BACKGROUND: Metabolic syndrome (MetS) facilitates the development of cardiovascular disease due to atherosclerosis, which is accelerated by defects of the vascular endothelium. Vascular dysfunction in response to nitric oxide (NO) occurs in the mesenteric arteries of an animal model of MetS, SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats. Vascular responses to vasodilators are affected by perivascular adipose tissue (PVAT) that surrounds the outsides of arteries. In this study, we assessed the role of PVAT in vascular dysfunction observed in SHRSP.ZF. METHODS: To determine the effects of PVAT on vasodilators in SHRSP.ZF and control Wistar-Kyoto (WKY) rats, we used organ bath bioassay techniques to assay acetylcholine and nitroprusside-induced relaxations of isolated mesenteric arterial ring preparations with PVAT intact or removed. RESULTS: A PVAT-mediated enhancement of relaxations induced by acetylcholine and nitroprusside occurred in SHRSP.ZF at 20 weeks of age, but not at 10 and 30 weeks, and did not occur in WKY. Furthermore, the enhancing effects of PVAT from SHRSP.ZF at 20 weeks could not be substituted by replacement with PVAT from either WKY or 30-week-old SHRSP.ZF, was inhibited by NO synthase inhibitor, and abolished by removal of the arteries' endothelium. Cyclic guanosine monophosphate (cGMP) accumulation elicited by nitroprusside was higher in SHRSP.ZF arteries with PVAT than arteries without PVAT at 20 weeks, but the enhancement of cGMP accumulation did not occur at 30 weeks. CONCLUSIONS: PVAT may regulate arterial tone by releasing diffusible vasorelaxing factor(s), which, through endothelium-derived NO production, compensates for impaired vasodilations at early stages of MetS.

Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2+ release system of Purkinje fibers in large mammalian heart.

BACKGROUND: Three distinct Ca2+ release channels were identified in dog P-cells: the ryanodine receptor subtype 2 (RyR2) was detected throughout the cell, while the ryanodine receptor subtype 3 (RyR3) and inositol phosphate sensitive Ca2+ release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca2+ cycling of P-cells is unclear. Recent modeling of Ca2+ mobilization in P-cells suggested that Ca2+ sensitivity of Ca2+induced Ca2+release (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca2+ release exists in live P-cells. We compared the regional Ca2+ dynamics with the arrangement of intracellular Ca2+ release (CR) channels. METHODS: Gene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca2+ activity and electrically-evoked Ca2+ mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca2+ events. We used the Ca2+ propagation velocities as indicators of channel Ca2+ sensitivity. RESULTS: RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP3R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca2+ release channels revealed an intermediate layer of RyR3 expression between a near-membrane InsP3R-region and a central RyR2-region. We found that cell periphery produced two distinct forms of spontaneous Ca2+-transients: (1) large asymmetrical Ca2+ sparks under the membrane, and (2) typical Ca2+-wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca2+ waves (CWWs) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3µM) and xestospongin C (XeC; 50µM) dramatically reduced their frequency. The Ca2+ wavelets propagated in a 5-10µm thick layered space which matched the intermediate zone of RyR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RyR3 antagonist dantrolene (10µM). The velocity of wavelets was two times larger (86±16µm/s; n=14) compared to CWWs' (46±10µm/s; n=11; P<0.05). Electric stimulation triggered a uniform and large elevation of Ca2+ concentration under the membrane which preceded the propagation of Ca2+ into the interior of the cell. Elevated Cai propagated at 150µm/s (147±34µm/s; n=5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75±24µm/s; n=5) in the central region wherein predominant RyR2 expression was detected. CONCLUSION: We identified two layers of distinct Ca2+ release channels in the periphery of Pcell: an outer layer of InsP3Rs under the membrane and an inner layer of RyR3s. The propagation of Ca2+ events in these layers revealed that Ca2+ sensitivity of Ca2+ release was larger in the RyR3 layer compared to that of other sub-cellular regions. We propose that RyR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers.

Age-related changes to vascular protease-activated receptor 2 in metabolic syndrome: a relationship between oxidative stress, receptor expression, and endothelium-dependent vasodilation.

Protease-activated receptor 2 (PAR2) is expressed in vascular endothelium. Nitric oxide (NO) - cyclic GMP-mediated vasodilation in response to 2-furoyl-LIGRLO-amide (2fLIGRLO), a PAR2-activating peptide, is impaired in aortas from aged SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome. Here we investigated mechanisms linking PAR2's vascular effects to phenotypic characteristics of male SHRSP.ZF rats at 10, 20, and 30 weeks of age. We found vasodilation responses to either 2fLIGRLO or enzyme-mediated PAR2 activation by trypsin were sustained until 20 weeks and lessened at 30 weeks. PAR2 protein and mRNA levels were lower in aortas at 30 weeks than at 10 and 20 weeks. PAR2-mediated responses positively correlated with PAR2 protein and mRNA levels. Decreased cGMP accumulation in the presence of 2fLIGRLO paralleled the decreased relaxations elicited by nitroprusside and the cGMP analog 8-pCPT-cGMP, and the less soluble guanylyl cyclase protein at 30 weeks. 2fLIGRLO-induced relaxation was negatively correlated with serum thiobarbituric acid reactive substances, an index of oxidative stress, which increased with age. Forward stepwise data regression supported a model of age-related decreases in PAR2 function resulting from decreased PAR2 mRNA and increased oxidative stress. We conclude that decreased responsiveness of aortic smooth muscle to NO and downregulation of receptor expression impair PAR2 functions at later stages of metabolic syndrome in SHRSP.ZF rats.

Characterization and Functions of Protease-Activated Receptor 2 in Obesity, Diabetes, and Metabolic Syndrome: A Systematic Review.

Proteinase-activated receptor 2 (PAR2) is a cell surface receptor activated by serine proteinases or specific synthetic compounds. Interest in PAR2 as a pharmaceutical target for various diseases is increasing. Here we asked two questions relevant to endothelial dysfunction and diabetes: How is PAR2 function affected in blood vessels? What role does PAR2 have in promoting obesity, diabetes, and/or metabolic syndrome, specifically via the endothelium and adipose tissues? We conducted a systematic review of the published literature in PubMed and Scopus (July 2015; search terms: par2, par-2, f2lr1, adipose, obesity, diabetes, and metabolic syndrome). Seven studies focused on PAR2 and vascular function. The obesity, diabetes, or metabolic syndrome animal models differed amongst studies, but each reported that PAR2-mediated vasodilator actions were preserved in the face of endothelial dysfunction. The remaining studies focused on nonvascular functions and provided evidence supporting the concept that PAR2 activation promoted obesity. Key studies showed that PAR2 activation regulated cellular metabolism, and PAR2 antagonists inhibited adipose gain and metabolic dysfunction in rats. We conclude that PAR2 antagonists for treatment of obesity indeed show early promise as a therapeutic strategy; however, endothelial-specific PAR2 functions, which may offset mechanisms that produce vascular dysfunction in diabetes, warrant additional study.

Small caliber arterial endothelial cells calcium signals elicited by PAR2 are preserved from endothelial dysfunction.

Endothelial cell (EC)-dependent vasodilation by proteinase-activated receptor 2 (PAR2) is preserved in small caliber arteries in disease states where vasodilation by muscarinic receptors is decreased. In this study, we identified and characterized the PAR2-mediated intracellular calcium (Ca(2+))-release mechanisms in EC from small caliber arteries in healthy and diseased states. Mesenteric arterial EC were isolated from PAR2 wild-type (WT) and null mice, after saline (controls) or angiotensin II (AngII) infusion, for imaging intracellular calcium and characterizing the calcium-release system by immunofluorescence. EC Ca(2+) signals comprised two forms of Ca(2+)-release events that had distinct spatial-temporal properties and occurred near either the plasmalemma (peripheral) or center of EC. In healthy EC, PAR2-dependent increases in the densities and firing rates of both forms of Ca(2+)-release were abolished by inositol 1,4,5- trisphosphate receptor (IP3R) inhibitor, but partially reduced by transient potential vanilloid channels inhibitor ruthenium red (RR). Acetylcholine (ACh)-induced less overall Ca(2+)-release than PAR2 activation, but enhanced selectively the incidence of central events. PAR2-dependent Ca(2+)-activity, inhibitors sensitivities, IP3R, small- and intermediate-conductance Ca(2+)-activated potassium channels expressions were unchanged in EC from AngII WT. However, the same cells exhibited decreases in ACh-induced Ca(2+)-release, RR sensitivity, and endothelial nitric oxide synthase expression, indicating AngII-induced dysfunction was differentiated by receptor, Ca(2+)-release, and downstream targets of EC activation. We conclude that PAR2 and muscarinic receptors selectively elicit two elementary Ca(2+) signals in single EC. PAR2-selective IP3R-dependent peripheral Ca(2+)-release mechanisms are identical between healthy and diseased states. Further study of PAR2-selective Ca(2+)-release for eliciting pathological and/or normal EC functions is warranted.