Endothelium-dependent Acetylcholine Research Articles

Dual Strategy with Adipose-Derived Stem Cells and l-arginine Recovered Cavernosal Functions in a Rat Model of Radical Prostatectomy.

As standard therapy for prostate cancer, radical prostatectomy causes cavernous nerve (CN) injury and increases fibrosis and hypoxia-induced penile structural alterations. This study aimed to determine the potential beneficial effects of adipose-derived stem cells (ADSCs) and l-arginine alone or in combination on the penile erection in a rat model of erectile dysfunction caused by bilateral cavernous nerve transection (CNT). Male rats (n = 35) were randomized into five groups: Sham-operated; CNT (4-weeks); CNT plus ADSCs (1 × 106 cells by intracavernosal injection); CNT plus l-arginine (4 weeks, 10 mg/kg/day, oral); and ADSCs combined with l-arginine in CNT. In vivo erectile responses and in vitro relaxant responses were measured. Western blot and immunohistochemistry analyses were used to determine the expression and localization of endothelial nitric oxide synthase, neuronal nitric oxide synthase, transforming growth factor-beta 1, hypoxia-inducible factor-1 alpha (HIF-1α), and apoptosis markers (Bax and Bcl-2). The ratio of smooth muscle to collagen and nerve regeneration were calculated using Masson's trichrome and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. The combined treatment restored diminished erectile responses, endothelium-dependent acetylcholine, and electrical field stimulation-induced relaxation of the corpus cavernosum in rats with CNT, whereas either monotherapy produced only partial improvements. All treatment regimens restored increases in the protein expression of HIF-1 and Bax in rats with CNT. The decrease in smooth muscle mass and NADPH-diaphorase-positive nerve fibers was partially ameliorated by monotherapy, whereas combined therapy led to recovery. These findings indicate that combined treatment with ADSCs and l-arginine may restore erectile function in rats with CNT by inhibiting hypoxia-induced neurotoxicity and preserving endothelium function and smooth muscle content.

Perivascular adipose tissue: a driver of sex-specific differences in the vascular outcomes of obesity

Obesity is a potent predictor of cardiovascular disease (CVD) and associated risk factors, including hypertension. Perivascular adipose tissue (PVAT) surrounds most blood vessels and modulates vascular tone. Under physiological conditions, PVAT exerts anti-contractile effects and sex differences are apparent in PVAT function. Under obese conditions, PVAT becomes dysregulated which may contribute to vascular dysfunction; how sex impacts the remodelling of PVAT and altered vascular contractility during obesity is unclear. We hypothesise that sex specific PVAT dysfunction in the setting of obesity is an important driver of sex differences in vascular pathologies and CVD risk.Adult (8wk) male and female C57Bl/6J mice received either chow diet (CD; 2.5%kcal fat) or high fat diet (HFD; 58%kcal fat) for 16 weeks (n=8/sex and diet). At 15 weeks of diet, blood pressure was assessed via tail-cuff plethysmography. At 16 weeks of diet, mesenteric arteries were isolated, and PVAT left intact or removed. Wire myography was used to assess the dilatory response to endothelium-dependent acetylcholine (ACh) and endothelium independent sodium nitroprusside (SNP). LogEC50 and %Preconstriction were calculated for each concentration response curve. PVAT was dissected from mesenteric arteries (mPVAT) and flash frozen for bulk RNA sequencing or fixed for histology. Data are mean ± SD and were analysed by two-way ANOVA.Under CD, the removal of PVAT did not change vasodilatory response to ACh or SNP in either sex. Under HFD, male mice showed reduced sensitivity (logEC50 PVAT vs no PVAT, p=0.0349) and maximal dilatory response (%Preconstriction PVAT vs no PVAT, p=0.0119) to ACh. This effect was not observed in females. Moreover, PVAT exerted no effect on dilatory responses to SNP in HFD groups of either sex, indicating an endothelium-dependent effect. Blood pressure was not altered in either sex by HFD. Transcriptomic analysis using RNA sequencing revealed female mPVAT is clearly distinguished from male mPVAT under HFD, but not CD conditions, with significantly down-regulated extracellular matrix remodelling and pro-inflammatory pathways, and significantly upregulated oxidative phosphorylation pathways. Histological analysis revealed potentiated adipocyte hypertrophy and collagen staining in mPVAT from males compared to females under HFD (p=0.034 and p=0.018 respectively).Our study shows that female mice are more protected from adverse effects of HFD, including PVAT-mediated endothelial dysfunction. Our RNA sequencing data provides insights into how pathophysiologic remodelling of mPVAT may contribute to the vascular outcomes of obesity-associated hypertension and suggests that this may be modulated by sex hormones. Further studies will unpick the underlying mechanism to identify potential targets for therapeutic intervention in obesity-associated hypertension. Funded by British Heart Foundation grant FS/19/55/34890. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GLUCOCORTICOID EXCESS INDUCES NON-DIPPING BLOOD PRESSURE AND RENAL VASCULAR DYSFUNCTION

Abstract Objective: Blood pressure (BP) abnormalities such as loss/attenuation of nocturnal BP dip (non-dipping) increase cardiovascular risk. The mechanisms underlying non-dipping are not clear, but renal and vascular dysfunction has been implicated. People with abnormal glucocorticoid (GC) homeostasis, such as in Cushing's syndrome, often display non-dipping BP therefore, here we investigated the contribution of GCs to BP rhythmicity. Design and method: The effect of GC excess, driven by ACTH infusion, on BP and plasma GC rhythmicity was examined in adult male C57BL/6J mice. Radiotelemetry was used to measure BP longitudinally before and after ACTH infusion (2.5 mg/day for 14days via minipump). Diurnal plasma GCs was measured by ELISA. Diurnal renal vascular function was investigated ex vivo by wire myography in isolated renal arteries from separate cohorts of mice either control or ACTH-treated collected between 7–8AM (day) or 7–8PM (night). Vascular reactivity to vasoconstrictor phenylephrine (PHE), endothelium-dependent acetylcholine (ACh) and endothelium-independent sodium nitroprusside (SNP) vasodilators was assessed. Data are mean ± SD. Results: After 5days of baseline measurements, 24-hour systolic BP (SBP) was 123.8 ± 4.2 mmHg and 24-hour diastolic BP (DBP) was 96.2 ± 5.6 mmHg. The day:night SBP and DBP variation was 12.8 ± 4.5 mmHg and 12.3 ± 4.8 mmHg, respectively. ACTH-excess increased 24-hour SBP to 133.8 ± 1.7 mmHg and 24-hour DBP to 101.4 ± 2.1 mmHg. The day:night SBP and DBP variation was reduced to 0.6 ± 7.2 mmHg (p = 0.0166) and 2.4 ± 9.6 mmHg (p = 0.0235), respectively. Day:night activity count during BP measurements was similar before and after ACTH infusion. Diurnal plasma GCs was increased following ACTH infusion, compared with baseline (p < 0.0001). Furthermore, ACTH-excess flipped the diurnal rhythm of GC, so the day plasma GC concentration is higher than the night. Day control renal arteries showed a trend for an increase in sensitivity to PHE (p = 0.0578) and SNP (p = 0.0795), with no changes in ACh (p = 0.1299), compared with night control renal arteries. ACTH-excess caused no differences in day and night responses to PHE (p = 0.5526), ACh (p = 0.1169), and SNP (p = 0.9466). ACTH-excess caused renal vascular dysfunction, compared with controls, causing a significant reduction in sensitivity to PHE (p = 0.0005), ACh (p = 0.0128) and SNP (p = 0.0002). Conclusions: These data show that chronic GC induces non-dipping BP and renal vascular dysfunction.

Myeloid GRK2 Regulates Obesity-Induced Endothelial Dysfunction by Modulating Inflammatory Responses in Perivascular Adipose Tissue.

Perivascular adipose tissue (PVAT) is increasingly being regarded as an important endocrine organ that directly impacts vessel function, structure, and contractility in obesity-associated diseases. We uncover here a role for myeloid G protein-coupled receptor kinase 2 (GRK2) in the modulation of PVAT-dependent vasodilation responses. GRK2 expression positively correlates with myeloid- (CD68) and lymphoid-specific (CD3, CD4, and CD8) markers and with leptin in PVAT from patients with abdominal aortic aneurysms. Using mice hemizygous for GRK2 in the myeloid lineage (LysM-GRK2+/−), we found that GRK2 deficiency in myeloid cells allows animals to preserve the endothelium-dependent acetylcholine or insulin-induced relaxation, which is otherwise impaired by PVAT, in arteries of animals fed a high fat diet (HFD). Downregulation of GRK2 in myeloid cells attenuates HFD-dependent infiltration of macrophages and T lymphocytes in PVAT, as well as the induction of tumor necrosis factor-α (TNFα) and NADPH oxidase (Nox)1 expression, whereas blocking TNFα or Nox pathways by pharmacological means can rescue the impaired vasodilator responses to insulin in arteries with PVAT from HFD-fed animals. Our results suggest that myeloid GRK2 could be a potential therapeutic target in the development of endothelial dysfunction induced by PVAT in the context of obesity.

The Protective Mechanism Of Exercise Training For Coronary Vascular Dysfunction In Atherosclerosis: Er Stress And Ucp-2

PURPOSE: Endoplasmic reticulum (ER) stress and uncoupling protein-2 (UCP-2) are a key modulator for endothelial dysfunction in atherosclerosis. We determined the effects of exercise on ER stress and UCP-2 in endothelial dysfunction in atherosclerosis. METHODS: We used 4 groups of mice; wild type (WT), WT with exercise training, running on the treadmill for 12 weeks (WT-EX), apolipoprotein E knockout (ApoE KO) and ApoE KO with exercise training (ApoE KO-EX). We measured endothelium-dependent acetylcholine (ACh)-induced vasodilation of isolated and pressurized coronary arterioles in a concentration-dependent manner. Also, ACh-induced vasodilation was elicited in the presence of an inhibitor of eNOS and UCP-2 (L-NAME and Genipin) and the ER stress inducer (Tunicamycin). Immunoblotting was performed to measure the protein expression of ER stress markers (GRP78, IRE1, eIF2α, and CHOP), NLRP3 inflammasome signaling (NLRP3, caspase-1, IL-1β), Bax, TXNIP, and UCP-2 in the heart. The expression of p67phox and superoxide were visualized using immunofluorescence and DHE staining in coronary arterioles. NO production was measured by nitrate/nitrite assay. RESULTS: ACh-induced endothelium-dependent vasodilation was attenuated in coronary arterioles of ApoE KO, but it was improved in ApoE KO-EX. L-NAME, tunicamycin, and Genipin attenuated vasodilation in WT, WT-EX and ApoE KO-EX, but not in ApoE KO. Exercise training reduced the expressions of ER stress (GRP78, p/t-IRE1, p-eIF2α, and CHOP), NLRP3 inflammasome (TXNIP, caspase-1 p20, and IL-1β), Bax, superoxide production, and NADPH oxidase p67phox, but it increased NO production in ApoE KO-EX mice. CONCLUSION: Our findings suggest that exercise training alleviates endothelial dysfunction in atherosclerotic coronary arterioles through the NOS, UCP-2, and ER stress signaling pathways including TXNIP/NLRP3 inflammasome and oxidative stress.

P2709Wnt5a contributes to human atherosclerosis via novel USP17 redox signalling

Abstract Background Wnt5a is a non-canonical Wnt ligand with potential vascular effects, but its mechanistic role in atherosclerosis progression and the underlying downstream mechanisms are poorly explored. Purpose To address the hypothesis that Wnt5a induces vascular NADPH-oxidases activity, endothelial dysfunction and detrimental downstream redox signalling which could propagate atherosclerosis in humans. Methods Study 1 included 70 patients with coronary artery disease (CAD) versus age- and sex-matched non-CAD controls. Study 2 included 1,003 CAD patients undergoing cardiac surgery; internal mammary artery (IMA) and saphenous vein (SV) segments were harvested and used for ex vivo experiments. Study 3 included 68 individuals undergoing two cardiac computed tomography scans 3–5 years apart; calcified plaque burden was assessed by coronary calcium score (CCS). Superoxide (O2·−) generation was measured by lucigenin chemiluminescence with NADPH 100μM stimulation as indicator of NADPH-oxidases activity. Activation of Rac1, a key NADPH-oxidases subunit, was evaluated by a commercially available kit. Primary vascular smooth muscle cells (VSMCs) and HeLa cells were used for in vitro experiments. Circulating Wnt5a and Sfrp5 (a Wnt5a antagonist) were measured by ELISA in fasting plasma samples. Results In Study 1, the presence of CAD was independently linked with increased circulating Wnt5a bioavailability (A), which was, in turn, associated with increased IMA NADPH-oxidases activity in Study 2 (B). Recombinant Wnt5a directly stimulated NADPH-oxidases activity (C) via Rac1 activation (not shown) in human IMA, while inducing endothelial dysfunction evidenced by impaired SV endothelium-dependent acetylcholine (Ach) vasorelaxations (D). Transcriptomic analysis in Wnt5a-treated primary VSMCs versus controls identified USP17, a deubiquitinating enzyme implicated in Rac1 activation, as the top differentially regulated hit (not shown). Indeed, Wnt5a stimulated USP17 upregulation in VSMCs which was reversed by PEGylated superoxide dismutase (peg-SOD) 300U/mL (E), suggesting a redox sensitive effect. USP17 knockdown abolished the ability of Wnt5a to induce Rac1 activation in HeLa cells (F). At a clinical level, plasma Wnt5a was a predictor of plaque progression (defined as ΔCCS≥1, G) and new onset calcification (H) in Study 3. Conclusions We demonstrate for the first time that Wnt5a is elevated in CAD and causally associated with increased vascular oxidative stress and endothelial dysfunction in humans. We further reveal USP17 to be a novel, previously undescribed, link between Wnt5a, Rac1 activation and NADPH-oxidase activity induction in humans. We finally propose that circulating Wnt5a may have a clinically relevant role in predicting atherosclerosis progression. Our findings identify Wnt5a as rational therapeutic target in vascular disease. Acknowledgement/Funding British Heart Foundation; Alexandros S Onassis Public Benefit Foundation

Testosterone Replacement Enhances Internal Pudendal Artery Relaxation to Reverse Erectile Dysfunction in a Rat Model of Androgen Deprivation Therapy

Introduction/ObjectivesAndrogen deprivation therapy (ADT) is frequently used to manage prostate cancer; however, erectile dysfunction (ED) and cardiovascular disease are common side effects. After cessation of ADT, testosterone (T) levels may take years to recover leaving men prone to cardiovascular disease and long‐term ED. The internal pudendal arteries (IPA) supply blood to the penis and vascular injury to these vessels can lead to ED. This study will determine if castration impairs vascular function in systemic (aorta and mesenteric arteries) and penile vasculature (IPA), and if T can restore erectile and vascular physiology. We hypothesize the IPA will develop impaired relaxation prior to systemic arteries following castration, and T therapy will recover both vascular and erectile function.MethodsMale Sprague Dawley rats (12 wks) were divided into 3 groups (n=8/grp): control (CON), CAST (6 wks castration), and CAST+T (6 wks castration+T (3mg/kg) in last 2 wks). Erections were assessed via cavernous nerve stimulation and measurement of intracavernosal to mean arterial pressures (ICP/MAP). Aortas, mesenteric arteries, and IPA were excised, cut (2 mm) and mounted into tissue baths. Contractility to high concentration potassium solution (KCl), electrical field stimulation (EFS), and phenylephrine (PE) were measured. Endothelial dependent acetylcholine (ACh) relaxation and endothelial independent relaxation to DEA NONOate were assessed. Non‐adrenergic non‐cholinergic (NANC) relaxation was evaluated by EFS in the presence of guanethidine and atropine. Serum testosterone levels were measured via ELISA. Androgen receptor protein expression was measured in IPA.ResultsCAST markedly impaired erectile function (ICP/MAP; CON: 0.83±0.03, CAST: 0.31±0.02; p<0.05) and decreased IPA ACh relaxation (CON: 50.4±3.5%, CAST: 31.2±2.3%; p<0.05). DEA NONOate relaxation was unchanged with CAST. CAST diminished IPA NANC relaxation (CON: 40.8±5.1%, CAST: 22.1±3.9%; p<0.05). However, CAST did not impact IPA contraction. Interestingly, CAST did not change vascular contraction or relaxation in aortas or mesenteric arteries. IPA androgen receptor expression decreased after CAST (CON: 1±0.17, CAST: 0.39±0.03; p<0.05). T supplementation restored serum T (T (ng/ml); CON: 4.2±0.8, CAST: 0.1±0.02, CAST+T: 32.9±3.2: p<0.05), erectile function (ICP/MAP; CAST+T 0.74±0.01 p<0.05) and improved ACh relaxation in IPA to values greater than CON (CAST+T: 63.9±2.9%; p<0.05). Similarly, CAST+T enhanced IPA NANC relaxation to levels greater than CON (CAST+T: 69.2±1.4%; p<0.05). T supplementation did not impact IPA contractions and had no effect on aortic or mesenteric vasoreactivity.ConclusionsCastration induces severe IPA vascular damage, yet systemic vasculature is unaffected. T therapy dramatically enhances IPA endothelial dependent and NANC‐mediated relaxation. Though T therapy safety in cancer survivors remains controversial, a growing body of literature is finding no correlation between T therapy and cancer progression. In certain populations of prostate cancer survivors, T replacement is warranted.Support or Funding InformationBSOM start‐up grant, SMSNA, AUAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The development of salt-sensitive hypertension regulated by PSGL-1 gene in mice

Background and objectiveChronic inflammatory is involved in the development of salt-sensitive hypertension and other cardiovascular diseases. PSGL-1 plays an important role in the inflammatory response.Methods and resultsIn this study, we used PSGL-1−/− and PSGL-1+/+ mice fed with high salt diet to measure the blood pressure, inflammatory response and vascular injury. We found that, in PSGL-1+/+ mice, high salt diet resulted in high blood pressure with the increased expression of serum inflammatory cytokines IL-6, IL-1β and TNFɑ, vascular injury markers MCP-1, ET-1, and VWF, and renal macrophages and T cells infiltration, and endothelium-dependent acetylcholine vasodilation dysfunction. However, the influence was not found in PSGL-1−/− mice. The deficiency of PSGL-1 prevented the increased adhesion of peripheral blood mononuclear cells to endothelial cells by high salt environment.ConclusionsOur results indicate that PSGL-1 is involved in the development of salt-sensitive hypertension via vascular inflammation and injury. The high salt induced inflammation may be initiated by leukocytes and endothelial cells adhesion through PSGL-1 binding with P-selectin or/and E-selectin.

Acute exhaustive rowing exercise reduces skin microvascular dilator function in young adult rowing athletes.

The effect of acute exhaustive exercise session on skin microvascular reactivity was assessed in professional rowers and sedentary subjects. A potential involvement of altered hemodynamic parameters and/or oxidative stress level in the regulation of skin microvascular blood flow by acute exercise were determined. Anthropometric, biochemical, and hemodynamic parameters were measured in 18 young healthy sedentary men and 20 professional rowers who underwent a single acute exercise session. Post-occlusive reactive hyperemia (PORH), endothelium-dependent acetylcholine (ACh), and endothelium-independent sodium nitroprusside (SNP) microvascular responses were assessed by laser Doppler flowmetry in skin microcirculation before and after acute exercise. Serum lipid peroxidation products and plasma antioxidant capacity were measured using spectrophotometry. At baseline, rowers had significantly lower diastolic blood pressure (DBP) and heart rate (HR), and higher stroke volume (SV), PORH, and endothelium-dependent vasodilation than sedentary. Acute exercise caused a significant increase in systolic blood pressure, DBP, HR, and SV and a decrease in total peripheral resistance in both groups. Acute exercise induced a significant impairment in PORH and ACh-induced response in rowers, but not in sedentary, whereas the SNP-induced vasodilation was not affected by acute exercise in any group. Antioxidant capacity significantly increased only in sedentary after acute exercise. Single acute exercise session impaired microvascular reactivity and endothelial function in rowers but not in sedentary, possibly due to (1) more rowing grades and higher exercise intensity achieved by rowers; (2) a higher increase in arterial pressure in rowers than in sedentary men; and (3) a lower antioxidant capacity in rowers.

Abstract P347: Retinol-binding Protein 7 (RBP7) is Required for PPARG-mediated Endothelial Protection via Adiponectin

PPARγ protects against endothelial dysfunction by regulation of unknown target genes. One such target, RBP7, an intracellular fatty acid-binding protein, exhibits endothelium-specific expression, but its effect on vascular function remain unknown. We hypothesize that RBP7 is endothelial protective. We examined vascular responses in basilar artery (pressurized myograph) of RBP7-knockout (KO) and wild type (WT) mice fed normal chow (ND) or high fat diet (HFD) for 8 wks. Endothelium-dependent acetylcholine (ACh)-induced relaxation was significantly impaired in HFD-fed KO mice (ACh, 100μM: 33±7% KO vs 83±10% WT, p<0.05), but not in ND-fed groups. This response was ameliorated by pre-incubation with superoxide scavenger tempol (1mM) or PEG-superoxide dismutase (100 U/ml). Mean arterial pressure (measured by radiotelemetry), body weight, hepatic steatosis, fasting glucose, glucose tolerance, and insulin sensitivity were similar in HFD-fed KO and WT mice. To identify targets downstream of RBP7, RNA-Sequencing was performed on carotid arteries from 8-week HFD-fed WT and KO mice as well as ND-fed age-matched littermates. Adiponectin (AdipoQ), a PPARγ target, was increased ~6-fold in HFD-fed WT mice, a response that was markedly blunted in KO mice. RNA sequencing was confirmed by qPCR. There was no difference in plasma AdipoQ. AdipoQ protein is expressed in endothelial cells of carotid arteries and its level of expression was increased in HFD-fed WT but not KO mice (AdipoQ/CD31: 1.14±0.1 WT-HFD vs 0.82±0.1 WT-ND, p<0.05; 0.79±0.1 KO-HFD vs 0.81±0.04 KO-ND). This led us to hypothesize that AdipoQ is involved in RBP7-mediated endothelial protection. Incubation of basilar artery with mouse full-length AdipoQ protein (5 μg/mL, 4 hours) significantly ameliorated endothelial dysfunction (ACh, 100 μM: 56±6% AdipoQ+KO vs 26±3% KO, p<0.05) and blunted carotid artery superoxide production in HFD-fed KO mice. AdipoQ also protects against endothelial dysfunction caused by subpressor Ang-II in KO mice. We conclude that RBP7 protects the endothelium from oxidative stress-induced dysfunction through an AdipoQ-dependent mechanism. Our evidence suggests RBP7 is an essential cofactor for activation of some PPARγ target genes in the endothelium.

Sufentanil attenuates impairment of the endothelium-dependent vasodilation induced by hypoxia-reoxygenation in the rat coronary artery.

Sufentanil has been used broadly in cardiac surgery, but the mechanisms by which it modulates coronary vascular tone after ischemia-reperfusion injury are largely unknown. Effects of sufentanil on coronary tone and on the relaxation of rat coronary arteries (CAs) in response to endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) relaxing agents in the presence of hypoxia-reoxygenation (H/R) was studied in an in vitro organ chamber setup. Sufentanil (10-7-10-4 mol/L) relaxed rat CA rings in endothelium-dependent and endothelium-independent manners. In endothelium-intact rings, preincubation of H/R-treated CAs with sufentanil (10-5 mol/L) significantly increased the acetylcholine response, but did not augment sodium nitroprusside-induced relaxation. Sufentanil-mediated potentiation of acetylcholine-induced relaxation was not affected by a nitric oxide synthase inhibitor or by intermediate- or small-conductance Ca2+-activated K+ channel blockers. However, potentiation was abolished by iberiotoxin (100 nmol/L), a selective inhibitor of large-conductance Ca2+-activated K+ channels, as well as Rp-cAMPS (30 μmol/L), a cyclic AMP-dependent protein kinase (PKA) inhibitor. Sufentanil induced endothelium-dependent and endothelium-independent relaxation and attenuated H/R-induced impairment of endothelium-dependent vasodilation in the rat CAs. The potentiating effect of sufentanil may involve activation of large-conductance Ca2+-activated K+ channels via cAMP-dependent mechanisms.

Abstract MP15: Afferent Arteriolar Endothelial-dependent Dilation is Impaired Prior to the Development of Radiation-induced Nephropathy and Hypertension

Chronic kidney disease (CKD) occurs in 15% of hematopoietic stem cell transplant (HSCT) patients, and has been clearly linked to irradiation at the time of the HSCT. CKD due to radiation also occurs after radionuclide therapy for cancer. Radiation nephropathy is expressed in rats and in humans as proteinuria, azotemia, and hypertension. There is a latent period of 6-8 weeks after irradiation to the development of proteinuria, azotemia, and hypertension in rats. This study tested the hypothesis that afferent arteriolar responses to the endothelial-dependent dilator acetylcholine are impaired prior to the development of proteinuria, azotemia, and hypertension in rats exposed to total body irradiation (TBI). Male WAG/RijCmcr rats were subjected to TBI (11 Gy) and afferent arteriolar responses to acetylcholine using the juxtamedullary nephron technique were determined at one, three, and six weeks. Systolic blood pressure (117 ± 6 vs. 119 ± 4 mmHg) and BUN (15.6 ± 1.4 vs. 15.8 ± 0.8 mg/dl) were not different between control and TBI groups at 6 weeks. Afferent arteriolar diameters averaged 22.5 ± 0.8 μm (n=30) in controls and 21.7 ± 0.7 μm (n=27) in TBI rats and were not different between control and TBI groups at 1, 3, or 6 weeks. Acetylcholine dilator responses were progressively attenuated from one to six weeks in TBI compared to control rats. During exposure to acetylcholine (0.01, 0.1, 1, and 10 μmol/L), afferent diameter increased by 8 ± 2%, 18 ± 3%, 27 ± 2% and 31 ± 3% in control rats (n=6), and 3 ± 2%, 9 ± 3%, 12 ± 3%, and 16 ± 3% in kidneys of 3 week TBI rats (n=8, p<0.05). However, TBI did not change the norepinephrine-mediated vasoconstrictor responses or the nitroprusside vasodilator responses at 1, 3, or 6 weeks. Renal microvessels were isolated from control and TBI groups at 3 and 6 weeks for protein expression assessment of endothelial enzymatic pathways Cyp2C, COX, and NOS. We found that epoxygenase Cyp2C23 and Cyp2C11 protein expressions were significantly lower 3 and 6 weeks following TBI compared to control rats. Taken together, these results indicate that the impaired afferent arteriolar endothelial-dependent acetylcholine responses and decreased epoxygenase enzymes precede the development of proteinuria, azotemia, and hypertension in irradiated rats.

Effects of different frequencies of transcutaneous electrical nerve stimulation on venous vascular reactivity.

Transcutaneous electrical nerve stimulation (TENS) is a type of therapy used primarily for analgesia, but also presents changes in the cardiovascular system responses; its effects are dependent upon application parameters. Alterations to the cardiovascular system suggest that TENS may modify venous vascular response. The objective of this study was to evaluate the effects of TENS at different frequencies (10 and 100 Hz) on venous vascular reactivity in healthy subjects. Twenty-nine healthy male volunteers were randomized into three groups: placebo (n=10), low-frequency TENS (10 Hz, n=9) and high-frequency TENS (100 Hz, n=10). TENS was applied for 30 min in the nervous plexus trajectory from the superior member (from cervical to dorsal region of the fist) at low (10 Hz/200 μs) and high frequency (100 Hz/200 μs) with its intensity adjusted below the motor threshold and intensified every 5 min, intending to avoid accommodation. Venous vascular reactivity in response to phenylephrine, acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) was assessed by the dorsal hand vein technique. The phenylephrine effective dose to achieve 70% vasoconstriction was reduced 53% (P<0.01) using low-frequency TENS (10 Hz), while in high-frequency stimulation (100 Hz), a 47% increased dose was needed (P<0.01). The endothelium-dependent (acetylcholine) and independent (sodium nitroprusside) responses were not modified by TENS, which modifies venous responsiveness, and increases the low-frequency sensitivity of α1-adrenergic receptors and shows high-frequency opposite effects. These changes represent an important vascular effect caused by TENS with implications for hemodynamics, inflammation and analgesia.

Effects of exercise training and Mediterranean diet on vascular risk reduction in post-menopausal women

This study tested the potential risk-reduction benefits of Mediterranean Diet (MD) and regular exercise training on microvascular activity and cardiorespiratory capacity in postmenopausal women. Fifteen sedentary postmenopausal participants (age = 54.6 ± 3.6) were randomised into either exercise training or exercise combined with following MD for eight-weeks, and were assessed for their cardiorespiratory capacity, and upper- and lower-limb endothelial cutaneous vascular conductance (CVC) test using Laser Doppler Fluximetry (LDF), coupled with measuring endothelium-dependent Acetylcholine Chloride (Ach) and -dependent Sodium Nitropurruside (SNP) vasodilators. Exercise training improved cardiorespiratory capacity as indicated by ventilatory threshold (11.5 ± 2.1 vs. 14.0 ± 3.0 ml·kg-1·min-1, p < 0.05) and improved the microcirculatory perfusion results of CVC for both vasodilators Ach (p < 0.001, d = 0.65) and SNP (p = 0.003, d = 0.53) in the lower-limb and ACh (p = 0.01, d = 0.41) and SNP (p = 0.03, d = 0.48) in the upper-limb, all (p < 0.05). However, combining exercise with MD showed a stronger improvement in Ach (p = 0.02, d = 0.36) of the lower limb, than in exercise alone group. The results suggest that regular moderate exercise improves microcirculatory vascular function and increases exercise tolerance, both are responsible for reducing cardiovascular risk in postmenopausal women. However, combining MD with exercise suggests additional microvascular vasodialiatory improvement, suggesting an effective strategy for further cardiovascular risk-reduction in this high-risk group.

Short-Term Administration of Alibernet Red Wine Extract Failed To Affect Blood Pressure and To Improve Endothelial Function in Young Normotensive and Spontaneously Hypertensive Rats

As wine polyphenols were shown to possess many positive effects in mammals, including improvement of vascular function, this study investigated the effect of the Slovak Alibernet red wine extract (AWE) on blood pressure and vascular function in young normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Six weeks old, male, WKY and SHR were treated with AWE for three weeks at the dose of 24.2 mg/kg/day. Blood pressure (BP), determined by tail-cuff plethysmography, was significantly elevated in SHR vs. WKY and AWE failed to affect it. Lipid peroxidation was evaluated by determination of thiobarbituric acid-reactive substances. Vascular function was assessed in rings of the femoral artery using Mulvany-Halpern's myograph. Maximal endothelium-dependent acetylcholine (ACh)-induced relaxation was reduced in control SHR vs. WKY rats by approximately 9.3 %, which was associated with a significant decrease of its NO-independent component. AWE failed to affect maximal ACh-induced relaxation, both its NO-dependent and independent components, compared to controls of the same genotype. AWE however reduced lipid peroxidation in the left ventricle of both WKY and SHR and in the liver of SHR. In conclusion, three-week administration of AWE failed to reduce BP and to improve endothelial function in the femoral arteries of both genotypes investigated.

Prenatal Testosterone Induces Sex-Specific Dysfunction in Endothelium-Dependent Relaxation Pathways in Adult Male and Female Rats1

Prenatal testosterone (T) exposure impacts postnatal cardiovascular function, leading to increases in blood pressure with associated decreased endothelium-dependent vascular relaxation in adult females. Endothelial function in males is not known. Furthermore, which of the endothelial pathways contributes to endothelial dysfunction and if there exists sex differences are not known. The objective of this study was to characterize the relative contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the impaired endothelium-dependent vasodilation in prenatal T-exposed adult males and females. Offspring of pregnant rats treated with T propionate or its vehicle were examined. Telemetric blood pressure levels and endothelium-dependent vascular reactivity were assessed with wire myography. Levels of nitric oxide synthase (NOS3) and Kcnn3 and Kcnn4 channel expression were examined in mesenteric arteries. Mean arterial pressure was significantly higher in T males and females than in controls. Endothelium-dependent acetylcholine relaxation was significantly lower in both T males and females. EDHF-mediated relaxation was specifically blunted in T males (Emax = 48.64% ± 3.73%) compared to that in control males (Emax = 81.71% ± 3.18%); however, NO-mediated relaxation was specifically impaired in T females (Emax = 36.01% ± 4.29%) compared with that in control females (Emax = 54.56% ± 6.37%). Relaxation to sodium nitroprusside and levcromakalim were unaffected with T-treatment. NOS3 protein was decreased in T females but not in T males. Kcnn3 expression was decreased in both T males and females compared to controls. These findings suggest that prenatal T leads to an increase in blood pressure in the adult offspring, associated with blunting of endothelial cell-associated relaxation and that the effects are sex-specific: EDHF-related in males and NO-related in females.

OS056. Angiotensin II sensitivity and endothelial dysfunction afterexperimental preeclampsia

Women who suffered from preeclampsia (PE) have an increased risk for cardiovascular and renal diseases later in life. Although the exact mechanisms underlying this relationship are unknown, they may relate to an increased sensitivity to angiotensin II (Ang-II) and endothelial dysfunction during a preeclamptic pregnancy, which may persist after PE. Recently, we showed vascular hypersensitivity to Ang-II and disturbed endothelial cell function in experimental PE in rats as compared to healthy pregnant rats. To study whether vascular hypersensitivity to Ang-II and endothelial dysfunction persist postpartum in experimental PE. In this ongoing study, we thus far included non-pregnant rats (NP;n=9), formerly healthy pregnant rats (HP;n=9) and formerly experimental preeclamptic rats (PE; infusion of a low dose endotoxin; n=16). Six weeks after pregnancy, animals from each group were treated with Ang-II (osmotic minipump; 200ng/kg/min;NP: n=5;HP: n=6;PE: n=8) or were sham treated (NP: n=4;HP: n=3;PE: n=8). Blood pressure was measured in all rats one day before and weekly after Ang-II or sham treatment (for three weeks). At termination, the aortas of sham operated rats were obtained. Aortic rings (2mm) were mounted for isotonic measurement of vasotonus. Endothelium-dependent acetylcholine- (ACh) mediated vasodilation was studied in phenylephrine-preconstricted rings in the presence of vehicle, N(G)-nitro-L-arginine methyl ester, indomethacin or both, followed by full concentration response curves for ACh (10(-8)M-10(-4)M). Ang-II sensitivity was assessed by obtaining full concentration response curves (10(-10)M-10(-6)M). AT-1 and AT-2 receptor sensitivity was determined by administration of Ang-II in the presence of the AT-1 receptor blocker losartan, or the AT-2 receptor blocker PD123319. Our results indicate no difference in mean (±SD) systolic blood pressure (SBP) between the three groups six weeks after delivery (NP: 129(±7);HP: 127(±9);PE: 123(±10)mmHg;p=0.248). However, after three weeks of Ang-II treatment, a trend was found towards a stronger increase in SBP in PE rats as compared to HP rats (45.7(±18.9)% vs 63.4(±20.1)% respectively;p=0.081). Although we found no differences in in-vitro Ang-II sensitivity between the three groups, NP rats showed a trend towards an increased sensitivity of the AT-2 receptor to Ang-II compared to both groups of formerly pregnant rats. Total ACh-mediated endothelial relaxation was not different between the three groups. However, contribution of both NO and EDHF components to ACh-mediated relaxation seemed decreased in both groups of formerly pregnant rats as compared to the NP rats. These preliminary data suggest that healthy rats that suffered from preeclampsia during pregnancy have increased in-vivo sensitivity to Ang-II postpartum as compared to rats with an uncomplicated pregnancy. Whether these differences are related to in-vitro- changes in Ang-II sensitivity or changes in endothelial function remains to be established.

Time dependence for vascular effects of water-soluble components of diesel exhaust in atherosclerosis rats

ObjectiveAmbient particulate air pollution can aggravate cardiovascular disease by mechanisms suggested to involve translocation of particles to the bloodstream and impairment of endothelial function. Whether the direct impairment is time-dependent...

Effects of reactive oxygen species and neutrophils on endothelium-dependent relaxation of rat thoracic aorta.

Reactive oxygen species (ROS) are produced in different metabolic processes including the respiratory burst of neutrophils accompanying local inflammation. The aim of this study was to analyze the effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP)-activated neutrophils, isolated from the guinea pig peritoneal cavity, on isolated rings of a large (conduit) artery, the rat thoracic aorta. FMLP-activated neutrophils enhanced the basal tension increased by α1-adrenergic stimulation. In phenylephrine-precontracted aortae, they elicited marked contraction, while in noradrenaline-precontracted rat aortal rings they caused a biphasic response (contraction-relaxation). To eliminate interaction of activated neutrophils with catecholamines, in the subsequent experiments the basal tension was increased by KCl-induced depolarization. Activated neutrophils evoked a low-amplitude biphasic response (relaxation-contraction) on the KCl-induced contraction. Not only the acetylcholine- and A23187-induced relaxations but also the catalase sensitive hydrogen peroxide (H2O2) elicited contractions were endothelium-dependent. Even though the acetylcholine-induced relaxation was changed by activated neutrophils and by the ROS studied, their effects differed significantly, yet none of them did eliminate fully the endothelium-dependent acetylcholine relaxation. The effect of activated neutrophils resembled the effect of superoxide anion radical (O2 •–) produced by xanthine/xanthine oxidase (X/XO) and differed from the inhibitory effects of Fe2SO4/H2O2-produced hydroxyl radical (•OH) and H2O2. Thus O2 •– produced either by activated neutrophils or X/XO affected much less the endothelium-dependent acetylcholine-activated relaxation mechanisms than did •OH and H2O2. In the large (conduit) artery, the effects of activated neutrophils and various ROS (O2 •–, •OH and H2O2) seem to be more dependent on muscle tension than on endothelial mechanisms.

Effect of PAR2 in regulating TNF-α and NAD(P)H oxidase in coronary arterioles in type 2 diabetic mice

Protease-activated receptor-2 (PAR2) is expressed in endothelial cells and mediates endothelium-dependent vasodilation. We hypothesized that PAR2 regulates tumor necrosis factor-alpha (TNF-α)-induced coronary arteriolar dysfunction in type 2 diabetic (db/db) mice. To test this, coronary arterioles from WT control, db/db, db/db mice treated with PAR2 antagonist FSLLRY-NH₂ (db/db+FSLLRY-NH₂) and db/db mice null for TNF (db(TNF-)/db(TNF-)) were isolated and pressurized (60 cmH₂O) without flow. Although vasodilation to the endothelium-independent vasodilator sodium nitroprusside (SNP) was not different among WT, db/db, db/db+FSLLRY-NH₂ and db(TNF-)/db(TNF-), endothelium-dependent acetylcholine (ACh)- and flow-mediated vasodilation were impaired in db/db mice but were enhanced in db(TNF-)/db(TNF-) mice and db/db mice treated with PAR2 antagonist. NOS inhibitor N (G)-nitro-L-arginine-methyl ester (L-NAME) significantly reduced ACh-induced dilation in WT, db(TNF-)/db(TNF-) and db/db+FSLLRY-NH₂, but did not alter the vasodilation in db/db mice. In contrast, cyclooxygenase (COX) inhibitor indomethacin (Indo) did not alter ACh-induced vasodilation in these four groups of mice. PAR2-activating peptide (PAR2-AP, 2-Furoyl-LIGRLO-am)-induced dilation was higher in db/db mice than that in WT, db(TNF-)/db(TNF-) and db/db mice treated with PAR2 antagonist. These effects were abolished by denudation, or in the presence of L-NAME or Indo. Protein expressions of TNF-α, PAR2, gp91(phox) and p47(phox) in the heart and isolated coronary arterioles were higher in db/db mice compared to WT mice. Administration of PAR2 antagonist to db/db mice reduced protein expression of TNF-α, gp91(phox) and PAR2. Protein expression of gp91(phox) and p47(phox) was lower in db(TNF-)/db(TNF-) compared to db/db mice. These results indicate that PAR2 plays a pivotal role in endothelial dysfunction in type 2 diabetes by up-regulating the expression/production of TNF-α and activating NAD(P)H oxidase subunit p47(phox).