Tail-cuff Plethysmography Research Articles

Immune Activation Promotes Cardiac Remodeling in a Mouse Model of Takotsubo Cardiomyopathy

Takotsubo Cardiomyopathy (TCM) is an acute, life-threatening condition characterized by a drop in ejection fraction (EF) and apical ballooning of the left ventricle (LV). TCM is most frequently observed in women and is associated with an emotional or physical stressor. Importantly, with medical intervention, cardiac dysfunction is transient, and EF is often restored over weeks to months. Despite this, patients who experience TCM are at an increased risk of additional cardiovascular events and premature mortality. The precise etiology of TCM is unknown but a wave of excess circulating catecholamines is thought to be a trigger. Furthermore, the cause for increased cardiovascular risk post-TCM is unknown. The goal of this study is to understand how the immune system may contribute to TCM and the associated cardiovascular event risk. To induce TCM, mice were given a single dose of 200mg/kg isoproterenol (ISO), i.p. and cardiac function was assessed by conscious echocardiography (ECG). An initial study compared the effect of ISO between sexes (n=4-6). Though ISO induced a drop in EF in both sexes after 24 hours (p<0.001 compared to vehicle (Veh)), females exhibited lower EF (46.64±3.11%) than males (68.36±2.62%, p<0.0001). This mirrors the increased prevalence of clinically defined TCM in females in human populations. For this reason, female mice were used for the following experiments. A time course study demonstrated that EF begins to normalize by Day 3 (78.78±2.70 v 90.80±1.00%, p<0.001) and is restored to baseline levels by Day 7 (90.65±1.17%). Flow cytometry was used to identify changes in immune cell populations in the heart on Days 0, 1, 3 and 7 (n=4-5). Neutrophils and inflammatory monocytes increased on Day 1, returning to baseline by Day 3. Dendritic cells and macrophages were elevated on Day 3 post-ISO, with a specific increase in CCR2+ TIM4- recruited inflammatory macrophages (p<0.0001). Across all subsets, the number of immune cells returned to baseline levels by Day 7. Due to the robust immune response observed, we tested the specific hypothesis that transfer of immune cells from mice who have experienced TCM would induce cardiac dysfunction in naïve recipient mice. Donor mice were treated with either Veh or ISO and 1 day later splenocyte isolation and transfer was performed. Importantly, transfer of splenocytes from mice who had TCM induced a significant drop in EF the following day in the recipient mice (86.30±1.08%, p=0.001, n=10) concurrent with an increase in LV inner diameter and volume during systole. A final study tested the hypothesis that a history of TCM would increase risk for future cardiovascular disease. Veh or ISO-treated mice were allowed to recover for 10 days. Tail-cuff plethysmography showed no significant difference in baseline blood pressure and minipumps were implanted to deliver a constant infusion of Angiotensin II (Ang II, 140ng/kg/min). Mice with a history of TCM exhibited higher blood pressures compared to controls (135.11±3.32 v 116.9±6.41mmhg, n=6/9, p<0.05) by week 2 of Ang II infusion. These results together demonstrated that TCM is associated with a robust immune response and points to a possible causal contribution of immune activation to the cardiac dysfunction observed in TCM. Furthermore, a history of TCM promotes a hypertensive phenotype, but additional studies are needed to test whether this is through immunological mechanisms. AHAPOST837441 DJF, 19EIA34480023-MSM, and 1R01 HL161212-MSM. This is the full abstract presented at the American Physiology Summit 2024 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.

Estrone-mediated lowering of ROS and NOX4 improves endothelial function in ovariectomized wistar rats.

Estrone (E1) constitutes the primary component in oral conjugated equine estrogens (CEEs) and serves as the principal estrogen precursor in the female circulation in the post-menopause. E1 induces endothelium-dependent vasodilation and activate PI3K/NO/cGMP signaling. To assess whether E1 mitigates vascular dysfunction associated with postmenopause and explore the underlying mechanisms, we examined the vascular effects of E1 in ovariectomized (OVX) rats, a postmenopausal experimental model. Blood pressure was measured using tail-cuff plethysmography, and aortic rings were isolated to assess responses to phenylephrine, acetylcholine (ACh), and sodium nitroprusside. Responses to ACh in rings pre-incubated with superoxide dismutase (SOD), catalase (CAT), or apocynin were also evaluated. Protein expression of SOD, CAT, NOX1, NOX2, and NOX4 was determined by Western blotting. E1 treatment resulted in decreased body weight and retroperitoneal fat, increased uterine weight, and prevented elevated blood pressure in the OVX group. Furthermore, E1 improved endothelium-dependent ACh vasodilation, activated compensatory antioxidant mechanisms - i.e. increased SOD and CAT antioxidant enzymes activity, and decreased NOX4 expression. This, in turn, helped prevent oxidative stress and endothelial dysfunction in OVX rats. Additionally, E1 treatment reversed the increased total LDL cholesterol observed in the OVX group. The findings underscore protective effects of E1 on the cardiovascular system, counteracting OVX-related oxidative stress and endothelial dysfunction in Wistar rats. E1 exhibits promising therapeutic benefits for managing cardiovascular health, particularly in postmenopausal conditions.

Chronic exercise improves renal AT1 and ETB receptor functions via modulating GRK4 expression in obese Zucker rats

ABSTRACT Background Physical activity has profound benefits on health, especially in patients with cardiovascular and metabolic disease. Exercise training can reduce oxidative stress, improve renal function, and thus lower blood pressure. However, the effect of exercise training on angiotensin II type 1 receptors (AT1R) and endothelin subtype B receptors (ETBR)-mediated diuresis and natriuresis in obese Zucker rats is unclear. Methods Lean and obese Zucker rats were exercised or placed on a nonmoving treadmill for 8 weeks. Blood pressure was measured by tail-cuff plethysmography, and functions of AT1R and ETBR in the kidney were measured by natriuresis, respectively. Results Our data showed that exercise training improved glucose and lipid metabolism, renal function and sodium excretion in obese Zucker rats, accompanied by decreased oxidative stress and GRK4 expression in obese Zucker rats. Moreover, exercise training reduced the Candesartan-induced an increase in diuresis and natriuresis and increased ETBR agonists (BQ3020)-mediated diuresis and natriuresis in obese Zucker rats, which were associated with decreased renal AT1R expression and ETBR phosphorylation levels. Conclusions The results demonstrate that exercise training lowers blood pressure via improving renal AT1R and ETBR function through modulating GRK4 expression in Obese Zucker Rats and provides potentially effective targets for obesity-related hypertension.

Angiotensin-(1-7) Treatment Early in Life Prevents Cardiac Hypertrophy in Adult Hypertensive Rats.

Angiotensin (Ang)-(1-7) is a cardioprotective peptide of the renin-angiotensin system. Prepuberty has been considered as a later susceptible window of development, and stressful factors in this life phase can induce chronic diseases in adulthood. We aimed to investigate whether the treatment with Ang-(1-7) during the prepuberty could attenuate the development of hypertension and cardiac injury in adult spontaneously hypertensive rats (SHRs). SHRs were treated with Ang-(1-7) (24 μg/kg/h) from age 4 to 7 weeks. Systolic blood pressure was measured by tail-cuff plethysmography up to 17th week. Thereafter, echocardiography was performed, and the rats were euthanized for the collection of tissues and blood. Ang-(1-7) did not change the systolic blood pressure but reduced the septal and posterior wall thickness, and cardiomyocyte hypertrophy and fibrosis in SHR. In addition, Ang-(1-7) reduced the gene expression of atrial natriuretic peptide and brain natriuretic peptide, increased the metalloproteinase 9 expression, and reduced the extracellular signal-regulated kinases 1/2 phosphorylation. Ang-(1-7) also prevented the reduction of Mas receptor but did not change the protein expression of angiotensin-converting enzyme, angiotensin-converting enzyme 2, AT1, and AT2. The treatment with Ang-(1-7) decreased the malondialdehyde (MDA) levels and increased superoxide dismutase-1 and catalase activities and protein expression of catalase. Our findings demonstrate that the treatment of SHR with Ang-(1-7) for 3 weeks early in life promotes beneficial effects in the heart later in life, even without altering blood pressure, through mechanisms involving the reduction of oxidative stress and ERK1/2 phosphorylation. In addition, this study supports the prepuberty as an important programming window.

Depletion of follicular B cell-derived antibody secreting cells does not attenuate angiotensin II-induced hypertension or vascular compliance.

Marginal zone and follicular B cells are known to contribute to the development of angiotensin II-induced hypertension in mice, but the effector function(s) mediating this effect (e.g., antigen presentation, antibody secretion and/or cytokine production) are unknown. B cell differentiation into antibody secreting cells (ASCs) requires the transcription factor Blimp-1. Here, we studied mice with a Blimp-1 deficiency in follicular B cells to evaluate whether antibody secretion underlies the pro-hypertensive action of B cells. 10- to 14-week-old male follicular B cell Blimp-1 knockout (FoB-Blimp-1-KO) and floxed control mice were subcutaneously infused with angiotensin II (0.7 mg/kg/d) or vehicle (0.1% acetic acid in saline) for 28 days. BP was measured by tail-cuff plethysmography or radiotelemetry. Pulse wave velocity was measured by ultrasound. Aortic collagen was quantified by Masson's trichrome staining. Cell types and serum antibodies were quantified by flow cytometry and a bead-based multiplex assay, respectively. In control mice, angiotensin II modestly increased serum IgG3 levels and markedly increased BP, cardiac hypertrophy, aortic stiffening and fibrosis. FoB-Blimp-1-KO mice exhibited impaired IgG1, IgG2a and IgG3 production despite having comparable numbers of B cells and ASCs to control mice. Nevertheless, FoB-Blimp-1-KO mice still developed hypertension, cardiac hypertrophy, aortic stiffening and fibrosis following angiotensin II infusion. Inhibition of follicular B cell differentiation into ASCs did not protect against angiotensin II-induced hypertension or vascular compliance. Follicular B cell functions independent of their differentiation into ASCs and ability to produce high-affinity antibodies, or other B cell subtypes, are likely to be involved in angiotensin II-induced hypertension.

Calcitriol reverses age-related hypertension via downregulating renal AP1/AT1R pathway through regulating mitochondrial function

ABSTRACT Background The vitamin D level in the blood is associated with the incidence of hypertension. The present study investigated whether or not calcitriol, an active form of vitamin D, reverses age-related hypertension. Methods Young (3-month-old) and aged (12-month-old) C57BL/6 male mice were administered with or without calcitriol at 150 ng/kg per day by oral gavage for 8 weeks. Blood pressure was measured by tail-cuff plethysmography and telemetry, and superoxide production in renal tissue was assessed by fluorescence imaging, and the protein expression of AP1/AT1R signaling pathway was examined by Western blot. Results We showed that 24-hour renal sodium excretion was impaired and blood pressure was increased in aged mice, which was related to the enhancement of renal AT1R expression and function. In addition, the expression of transcription factor AP1 (a dimer of c-Fos and c-Jun) and the binding of AP1 to the AT1R promoter region was significantly enhanced, accompanied by decreased nuclear translocation of Nrf2, abnormal mitochondrial function including decreased ATP production, NAD+/NADH ratio and mtDNA copy numbers, and increased reactive oxygen species. Calcitriol increased 24-hour urinary sodium excretion and reduced blood pressure in aged mice. Mechanically, calcitriol increased the nuclear translocation of Nrf2, improved mitochondrial function, reduced AP1 binding ability to AT1R promoter, which reversed enhanced AT1R expression and function, and lowered blood pressure in aged mice. Conclusions Our findings indicated that calcitriol reversed age-related hypertension via downregulating renal AP1/AT1R pathway through regulating mitochondrial function. Thus, calcitriol may be a valuable therapeutic strategy for age-related hypertension.

Antihypertensive effect of the stem bark aqueous extract of Garcinia lucida Vesque (Clusiaceae) in L-NAME-treated rats: Contribution of endothelium-dependent and -independent vasorelaxation

Garcinia lucida is used in Cameroonian folk medicine to handle a variety of ailments, including arterial hypertension. This study aimed at determining the phytochemical profile and the antihypertensive effect of the stem bark aqueous extract of G. lucida (AEGL). AEGL was subjected to LC-MS analysis, and its effect (75, 150, and 300 mg/kg/day; by gavage) was evaluated against Nω-nitro-L-arginine methyl ester (L-NAME; 40 mg/kg)-induced hypertension in adult male Wistar rats for four consecutive weeks. Blood pressure and heart rate were monitored weekly using tail-cuff plethysmography. The vasorelaxant effect of cumulative concentrations (3–10–30–100–300 μg/mL) of AEGL was examined on endothelium-intact and denuded thoracic aorta rings which were precontracted with KCl (90 mM) or norepinephrine (NE; 10−5 M), and in the absence or presence of L-NAME (10−4 M), indomethacin (10−5 M), methylene blue (10−6 M), tetraethylammonium (TEA, 5 × 10−6 M), glibenclamide (10 × 10−6 M) or propranolol (5 × 10−6 M). The influence of AEGL on the response to NE, KCl, and CaCl2 was also investigated. Six compounds, including Garcinia biflavonoids GB1 and GB2, were identified. AEGL prevented the development of hypertension (p < 0.01 and p < 0.001) without affecting the heart rate. AEGL induced a concentration-dependent relaxation of aortic rings precontracted with NE (EC50 = 7.915 μg/mL) that was significantly inhibited by the removal of the endothelium, L-NAME, or methylene blue (p < 0.05–0.001). Indomethacin, propranolol, TEA, and glibenclamide did not affect AEGL-evoked vasorelaxation. Preincubation of aortic rings with AEGL reduced the magnitude of contraction elicited by CaCl2 but did not alter that of KCl or NE. AEGL possesses an antihypertensive effect that is mediated by both endothelium-dependent and endothelium-independent mechanisms. The activation of the NO/sGC/cGMP pathway accounts for the endothelium-dependent vasorelaxation. These pharmacological effects of AEGL could be attributed to the presence of the Garcinia biflavonoids GB1 and GB2.

Abstract P233: Single-cell Transcriptomic Characterization Of Aortic Lymphocyte Heterogeneity During Hypertension

Vascular inflammation is a hallmark of hypertension. T and B lymphocytes accumulate in the aortic wall during hypertension; however, it is unclear which subtypes or functions contribute to disease progression. Thus, we aimed to characterize T and B cell subtypes, activation states and phenotypes in hypertension. Angiotensin II infusion (0.7 mg/kg/day; s.c. for 28 days, n = 15) was used to induce hypertension in 12-week-old male C57BL/6 mice. Age-matched normotensive controls received vehicle infusion (0.5% NaCl, 0.1% acetic acid; s.c. for 28 days, n = 11). Systolic blood pressure (SBP) was measured weekly by tail-cuff plethysmography. Aortic stiffness and fibrosis were assessed at endpoint via high resolution ultrasound and picrosirius red-staining, respectively. Whole aortas (n = 3 per treatment group) were isolated and digested into single-cell suspensions and enriched for live, nucleated, metabolically active cells. Single-cell RNA sequencing was performed using Chromium 10x and NovaSeq genomics platforms. Bioinformatic analyses identified cell types, acquired molecular and biological activities and inferred gene regulatory networks. At endpoint, angiotensin II-treated mice had elevated SBP (179 ± 4 vs. 124 ± 2 mmHg; P &lt; 0.0001), with increased aortic pulse wave velocity (1.5 ± 0.2 vs 3.1 ± 0.3 m/s; P = 0.001) and collagen content (51 ± 2% vs. 60 ± 2% wall area; P = 0.02), compared to vehicle controls. Total aortic T cells and B cells increased by 16% (184 vs. 215 cells) and 74% (433 vs. 771 cells) in hypertension, respectively. These increases were attributed to the expansion of CD8+, regulatory T cells (Tregs), gamma-delta (GD) T cells, immature transitional B2 cells, naïve B2, memory B2, B1 B cells and plasma cells, with a reduction in regulatory B2 B cells (Bregs). Gene ontology analyses revealed both pro- and anti-inflammatory B and T cells were activated during hypertension. However, antigen receptor activity pathways were only enriched in GD T cells, B1 B cells and effector Bregs (running enrichment scores &gt; 0.2). Overall, these findings identify GD T cells, B1 B cells and effector Bregs as key lymphocyte subtypes that drive vascular inflammation during experimental hypertension and aortic stiffening.

Cocoa Shell Extract Reduces Blood Pressure in Aged Hypertensive Rats via the Cardiovascular Upregulation of Endothelial Nitric Oxide Synthase and Nuclear Factor (Erythroid-Derived 2)-like 2 Protein Expression.

Cocoa shell is a by-product of cocoa manufacturing. We obtained an aqueous extract (CSE) rich in polyphenols and methylxanthines with antioxidant and vasodilatory properties. We aimed to evaluate the effects of CSE supplementation in aged hypertensive rats on blood pressure and the mechanism implicated. Eighteen-month-old male and female rats exposed to undernutrition during the fetal period who developed hypertension, with a milder form in females, were used (MUN rats). Systolic blood pressure (SBP; tail-cuff plethysmography) and a blood sample were obtained before (basal) and after CSE supplementation (250 mg/kg; 2 weeks, 5 days/week). Plasma SOD, catalase activity, GSH, carbonyls, and lipid peroxidation were assessed (spectrophotometry). In hearts and aortas from supplemented and non-supplemented age-matched rats, we evaluated the protein expression of SOD-2, catalase, HO-1, UCP-2, total and phosphorylated Nrf2 and e-NOS (Western blot), and aorta media thickness (confocal microscopy). MUN males had higher SBP compared with females, which was reduced via CSE supplementation with a significant difference for group, sex, and interaction effect. After supplementation with plasma, GSH, but not catalase or SOD, was elevated in males and females. Compared with non-supplemented rats, CSE-supplemented males and females exhibited increased aorta e-NOS and Nrf2 protein expression and cardiac phosphorylated-Nrf2, without changes in SOD-2, catalase, HO-1, or UCP-2 in cardiovascular tissues or aorta remodeling. In conclusion, CSE supplementation induces antihypertensive actions related to the upregulation of e-NOS and Nrf2 expression and GSH elevation and a possible direct antioxidant effect of CSE bioactive components. Two weeks of supplementation may be insufficient to increase antioxidant enzyme expression.

Type I collagen proteolysis by matrix metalloproteinase-2 contributes to focal adhesion kinase activation and vascular smooth muscle cell proliferation in the aorta in early hypertension

IntroductionIncreased matrix metalloproteinase (MMP)-2 activity contributes to increase vascular smooth muscle cell (VSMC) proliferation in the aorta in early hypertension by cleaving many proteins of the extracellular matrix. Cleaved products from type I collagen may activate focal adhesion kinases (FAK) that trigger migration and proliferation signals in VSMC. We therefore hypothesized that increased activity of MMP-2 proteolyzes type I collagen in aortas of hypertensive rats, and thereby, induces FAK activation, thus leading to increased VSMC proliferation and hypertrophic remodeling in early hypertension. MethodsMale Sprague-Dawley rats were submitted to renovascular hypertension by the two kidney-one clip (2K1C) model and treated with doxycycline (30 mg/kg/day) by gavage from the third to seventh-day post-surgery. Controls were submitted to sham surgery. Systolic blood pressure (SBP) was measured daily by tail-cuff plethysmography and the aortas were processed for zymography and Western blot for MMP-2, pFAK/FAK, integrins and type I collagen. Mass spectrometry, morphological analysis and Ki67 immunofluorescence were also done to identify collagen changes and VSMC proliferation. A7r5 cells were stimulated with collagen and treated with the MMP inhibitors (doxycycline or ARP-100), and with the FAK inhibitor PND1186 for 24 h. Cells were lysed and evaluated by Western blot for pFAK/FAK. Results2K1C rats developed elevated SBP in the first week as well as increased expression and activity of MMP-2 in the aorta (p < 0.05 vs. Sham). Treatment with doxycycline reduced both MMP activity and type I collagen proteolysis in aortas of 2K1C rats (p < 0.05). Increased pFAK/FAK and increased VSMC proliferation (p < 0.05 vs. Sham groups) were also seen in the aortas of 2K1C and doxycycline decreased both parameters (p < 0.05). Higher proliferation of VSMC contributed to hypertrophic remodeling as seen by increased media/lumen ratio and cross sectional area (p < 0.05 vs Sham groups). In cell culture, MMP-2 cleaves collagen, an effect reversed by MMP inhibitors (p < 0.05). Increased levels of pFAK/FAK were observed when collagen was added in the culture medium (p < 0.05 vs control) and MMP and FAK inhibitors reduced this effect. ConclusionsIncrease in MMP-2 activity proteolyzes type I collagen in the aortas of 2K1C rats and contributes to activate FAK and induces VSMC proliferation during the initial phase of hypertension.

Browning of perivascular adipose tissue prevents vascular dysfunction and reduces hypertension in angiotensin II-infused mice.

Hypertension is a world-leading cause of cardiovascular disease and premature deaths. Vascular tone is in part regulated by perivascular adipose tissue (PVAT) that releases pro and anticontractile factors. In hypertension, dysfunctional PVAT is observed and studies have indicated a causal relationship between dysfunctional PVAT and vascular damage in hypertension. The phenotype of PVAT on resistance vessels is primarily white adipose tissue. The present study investigates the impact of a changed phenotype, i.e., browning of PVAT, on vascular function and the development of hypertension. Browning was induced by β3-adrenergic agonist in control and angiotensin II-induced hypertensive mice. Studied parameters included blood pressure by tail-cuff plethysmography and vascular function by wire myography. Browning was confirmed through an immunohistochemical and gene analysis approach. The anticontractile effect of PVAT is lost in untreated hypertensive mice and vascular tone and blood pressure are increased. Browning of PVAT resulted in a maintained anticontractile effect, improved endothelial function, and reduced development of hypertension. Phenotype of PVAT is a major determinant of PVAT health during hypertensive conditions. Our data clearly demonstrates that browning of PVAT, i.e. changing the phenotype of PVAT, protects the vascular function and counteract the development of hypertension. This study provides novel insights into how PVAT can be protected in pathologies and thus limit the development of hypertension.

#5907 RENAL INJURY IN RELATION TO OBESITY AND THE ADDITIVE EFFECT OF HYPERTENSION IN FEMALE AND MALE OBESE AND LEAN ZSF1 RATS

Abstract Background and Aims Chronic kidney disease co-exists with heart failure with preserved ejection fraction (HFpEF), which is characterized by the presence of obesity, hypertension, and diabetes mellitus type 2. Obese ZSF1 rats, a model of HFpEF, exhibit multiple of these comorbidities that can disturb cardiac function. Little attention has been paid to how these comorbidities affect renal disease in the ZSF1 rats. HFpEF is found predominantly in women in whom obesity and hypertension are particularly prevalent. Therefore, we aimed to characterize the renal phenotype in female and male, lean and obese, ZSF1 rats and investigated additional effects of worsened hypertension on disease severity. Method Systolic blood pressure and renal function were assessed biweekly in female and male, lean and obese, ZSF1 rats from 12 to 26 weeks of age by tail-cuff plethysmography, urine collection via metabolic cages and plasma collection, respectively. From 19 weeks of age, rats were implanted with either a deoxycorticosterone acetate pellet (DOCA) and fed high salt diet (6% NaCl) or with a placebo pellet and fed a normal salt diet. At 26 weeks of age, terminal GFR was assessed via inulin clearance under isoflurane. Renal sections were processed for histological analysis. Results Lean and obese placebo ZSF1 rats, both female and male, were mildly hypertensive (systolic blood pressure 140-150 mmHg). All obese rats showed HFpEF, evidenced by elevated E/e’ ratio. In female normoglycemic ZSF1 rats, obesity associates with mild proteinuria, decreased GFR and glomerular hypertrophy. The addition of DOCA-salt worsened hypertension in female obese ZSF1 rats, but not in their lean counterparts. DOCA-salt-worsened hypertension enhanced proteinuria and triggered glomerulosclerosis in female obese rats. Male obese placebo ZSF1 rats were hyperglycemic and showed proteinuria, glomerular hypertrophy and sclerosis, and tubulo-interstitial damage. DOCA-salt worsened hypertension in both male lean and obese rats and further aggravated proteinuria, tubulo-interstitial damage, glomerular hypertrophy and sclerosis in obese male rats. No effect of DOCA-salt was observed on GFR in both male and female ZSF1 rats. Conclusion Female obese ZSF1 rats developed mild renal disease and DOCA-salt-worsened hypertension deteriorated renal function and structure in normoglycemic female obese ZSF1 rats similar to hyperglycemic male obese ZSF1 rats.

Proteasome inhibition reduces plasma cell and antibody secretion, but not angiotensin II-induced hypertension.

Depletion of mature B cells affords protection against experimental hypertension. However, whether B cell-mediated hypertension is dependent on differentiation into antibody-secreting cells (ASCs) remains unclear. Using the proteasome inhibitor, bortezomib, the present study tested the effect of ASC reduction on angiotensin II-induced hypertension. Male C57BL6/J mice were infused with angiotensin II (0.7 mg/kg/day; s.c.) for 28 days via osmotic minipump to induce hypertension. Normotensive control mice received saline infusion. Bortezomib (750 μg/kg) or vehicle (0.1% DMSO) was administered (i.v.) 3 days prior to minipump implantation, and twice weekly thereafter. Systolic blood pressure was measured weekly using tail-cuff plethysmography. Spleen and bone marrow B1 (CD19+B220-), B2 (B220+CD19+) and ASCs (CD138hiSca-1+Blimp-1+) were enumerated by flow cytometry. Serum immunoglobulins were quantified using a bead-based immunoassay. Bortezomib treatment reduced splenic ASCs by ∼68% and ∼64% compared to vehicle treatment in normotensive (2.00 ± 0.30 vs. 0.64 ± 0.15 × 105 cells; n = 10-11) and hypertensive mice (0.52 ± 0.11 vs. 0.14 ± 0.02 × 105 cells; n = 9-11), respectively. Bone marrow ASCs were also reduced by bortezomib in both normotensive (4.75 ± 1.53 vs. 1.71 ± 0.41 × 103 cells; n = 9-11) and hypertensive mice (4.12 ± 0.82 vs. 0.89 ± 0.18 × 103 cells; n = 9-11). Consistent with ASC reductions, bortezomib reduced serum IgM and IgG2a in all mice. Despite these reductions in ASCs and antibody levels, bortezomib did not affect angiotensin II-induced hypertension over 28 days (vehicle: 182 ± 4 mmHg vs. bortezomib: 177 ± 7 mmHg; n = 9-11). Reductions in ASCs and circulating IgG2a and IgM did not ameliorate experimental hypertension, suggesting other immunoglobulin isotypes or B cell effector functions may promote angiotensin II-induced hypertension.

Inhibition of endoplasmic reticulum stress restores the balance of renal RAS components and lowers blood pressure in the spontaneously hypertensive rats

ABSTRACT Background Endoplasmic reticulum (ER) stress has been shown to play a critical role in the pathogenesis of hypertension. However, the underlying mechanisms for lowering blood pressure (BP) by suppressing ER stress remain unclear. Here, we hypothesized that inhibition of ER stress could restore the balance between RAS components and lower BP in spontaneously hypertensive rats (SHRs). Methods Wistar-Kyoto (WKY) rats and SHRs received vehicle or 4-PBA, an ER stress inhibitor, in the drinking water for 4 weeks. BP was measured by tail-cuff plethysmography, and the expression of RAS components was examined by Western blot. Results Compared with vehicle-treated WKY rats, vehicle-treated SHRs exhibited higher blood pressure and increased renal ER stress and oxidative stress, accompanied by impaired diuresis and natriuresis. Moreover, SHRs had higher ACE and AT1R and lower AT2R, ACE2, and MasR expressions in the kidney. Interestingly, 4-PBA treatment improved impaired diuresis and natriuresis and lowered blood pressure in SHRs, accompanied by reducing ACE and AT1R protein expression and increasing AT2R, ACE2, and MasR expression in the kidneys of SHRs. In addition, these changes were associated with the reduction of ER stress and oxidative stress. Conclusions These results suggest that the imbalance of renal RAS components was associated with increased ER stress in SHRs. Inhibition of ER stress with 4-PBA reversed the imbalance of renal RAS components and restored the impaired diuresis and natriuresis, which, at least in part, explains the blood pressure-lowering effects of 4-PBA in hypertension.

Gender differences in phenotypic expressions of metabolic syndrome in ZSF rats

Aims: The focus of our laboratory has been to examine the mechanisms of insulin resistance and renal injury in metabolic syndrome. ZSF rats represent a transgenic murine model of human metabolic syndrome and spontaneously develop obesity, hyperglycemia, hypertension, progressive proteinuria, azotemia and severe hyperlipidemia. The renal and metabolic abnormalities in ZSF rats were first characterized and published from our laboratory (J Am Soc Neph 2007). However only male rats were examined in that study. Despite a totally similar genetic background, there could be significant gender differences in the expression of phenotypic characteristics of disease states. We examined the differences in the general and metabolic features of male and female rat models of metabolic syndrome. Methods: We studied male and female ZSF rats, an established model of metabolic syndrome. Obese ZSF (ZDFxSHHF-fa/facp) model was developed by crossing lean female Zucker Diabetic Fatty (ZDF ./fa) and lean male Spontaneously Hypertensive Heart Failure (SHHF/Mcc-facp, ./fa) rats. Male and female CD rats were used as controls. Rats were monitored from the 8th week of life and were fed normal rat chow (CD rats) or high calorie, high fat diet (Purina 5008) in the case of ZSF rats. The present report pertains to data obtained between 15-20 weeks of age. Blood pressure was measured by tail cuff plethysmography (Non-Invasive Blood Pressure measurements using the Kent CODA system). Blood and urine were examined for glucose (Ascensia Contour glucometer) and protein levels. Body weights were measured every week. Results: Our results show that at 20 weeks, males were heavier than the females in both ZSF (545±26g vs. 401±42g p&lt;0.05) and CD (608±73g vs. 301±22g p&lt;0.05) strains of rats. ZSF female rats were heavier than CD female rats. Male ZSF rats were fatter than male CD rats although the latter appear larger (no statistical difference in weights). There were no statistically significant differences in the BP amongst the four groups, which were all in the normal range. Blood glucose level were elevated in ZSF compared to CD rats. Male ZSF rats had higher glucose levels (342±36 mg/dl) compared to female ZSF rats (95±16mg/dl, p&lt;0.05). Urine glucose levels were higher in male ZSF rats while urine protein levels were higher in female ZSF rats compared to the other three groups. Conclusions: Although the male and female ZSF rats shared the same general genetic material, at 20 weeks of age they exhibited significant differences in various manifestations of metabolic syndrome with male ZSF rats exhibiting more advanced featues. These observations underscore the impact of gender and implied physiological factors in influencing the physical manifestations of a disease state. Private Research Endowment 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.

Chronic Unpredictable Stress Impairs Vascular Reactivity in Male and Female Mice

Background: Chronic stress is known to induce vascular inflammation causing endothelial and vascular smooth muscle dysfunction. Changes in the hypothalamic-pituitary-adrenal axis lead to increased sympathetic and decreased parasympathetic drive, causing the release of inflammatory cytokines in the vasculature, by mechanisms that are unclear. Hypothesis: We hypothesize that chronic unpredictable stress (CUS) increases reactive oxygen species and induces gasdermin pore formation and voltage-dependent anion channel (VDAC) oligomerization in the membrane of the mitochondria, which releases mitochondrial DNA (mtDNA) to the cytosol and induces TNF-α, IL-1β and IL-18 production through activation of the NLRP-3 inflammasome pathway. Methods: Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of chronic unpredictable stress (CUS) or handling (CTL). Systolic blood pressure (SBP) was measured at the end of the stress period by tail cuff plethysmography. Vascular function using myographs was assessed on isolated mesenteric resistance arteries (MRAs), pudendal artery (PA) and aorta from male and female CUS and CTL mice. The arterial rings were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2. Concentration-response curves to phenylephrine (PE; 1nM-30μM) and acetylcholine (ACh; 1nM-30μM) were performed. Expression of gasderminD was measured in the MRA by western blot. Concentration-response curves were analyzed using non-linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p&lt;0.05. Data were analyzed with GraphPad Prism 9.2.0. Results: SBP was increased by 12.1±4.9% in CUS mice. In CUS mice, compared to the CTL, the potency of ACh-induced relaxation was decreased in the MRA (pEC50 CUS 6.17±0.09 vs CTL 6.81±0.11; p=0.003) and PA (pEC50 CUS 6.65±0.07 vs CTL 6.31±0.09; p=0.008) but not in the aorta of male mice. In females, the potency to ACh was decreased only in the MRA (pEC50 CUS 5.94±0.30 vs CTL 6.98±0.15; p=0.041). There were no differences in the contractile responses to PE between controls and mice exposed to CUS in the MRA and PA of both males and females, while in the aorta of CUS males, there was a significant reduction in maximal response (EMAX CTL: 4.70±0.48 mN vs CUS 3.01± 0.23mN; p=0.007). The expression of gasderminD was increased in MRA of female mice only. Conclusion: Our results show that, in both sexes, CUS increased SBP and induced vascular dysfunction observed by impaired reactivity to ACh and PE, with increased expression of gasderminD in MRA of female mice only. These results suggest that the vascular adaptations to CUS are sex-dependent. This work was supported by NIH grants P20GM109091 (F.H.), R01HL130972-01A1 (F.G.S.), R01HL5949 (F.G.S.), RO1DK132948 (C.W. &amp; F.P.) and R01 MH129798 (SKW); VA grants: VISN7 RDA (F.H.), Merit awards: BX000168-10A1 (F.G.S.), BX005320 (F.G.S.), and BX002604 (M.J.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the abstract. 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.

A Kidney-Selective, Biopolymer-Delivered MMP-2 Inhibitory Peptide Protects Against Renal Injury in the Dahl Salt Sensitive Rat

Renal fibrosis, a hallmark of chronic kidney disease (CKD), is characterized by excessive deposition of extracellular matrix (ECM) components. Human and animal studies have shown that upregulation of the endopeptidase matrix metalloproteinase-2 (MMP-2) cleaves ECM and cytokines to promote inflammation and fibrosis in CKD. The objective of this study is to develop a novel, kidney-targeted MMP-2 inhibitor for prevention of renal fibrosis. Our group fused an MMP-2 inhibitory peptide (MMP-2i) to a kidney targeting biopolymer known as elastin like polypeptide (ELP). We previously optimized renal targeting of ELP-MMP-2i by testing the effect of ELP molecular weight on renal deposition and renal specificity, resulting in the selection of ELP 63 -MMP-2i (an ELP with 63 pentapeptide repeats and a molecular weight of 25 kDa) as a lead agent for continued therapeutic development. We also previously validated that ELP 63 -MMP-2i specifically inhibits MMP-2 without affecting other MMPs expressed in the kidney. The hypothesis of the current study is that our optimized agent, ELP 63 -MMP-2i, will ameliorate renal injury when administered to Dahl salt sensitive (Dahl S) rats fed a high-salt diet. To assess the efficacy of our therapeutic, 12-week-old Dahl S rats (male, n=4 per group) were fed a high salt (8% NaCl) diet and simultaneously dosed (300 nmol/kg/day s.c.) with either vehicle or ELP 63 -MMP-2i for 3 weeks. Metabolic cage housing was performed weekly to collect urine, blood pressure was assessed in conscious rats weekly by tail cuff plethysmography and at the experimental endpoint by direct measurement via an arterial catheter, and renal histology was assessed by Masson trichrome staining. Neither high-salt diet or ELP 63 -MMP-2i had any effect on the rats’ body weight. Blood pressure was elevated in Dahl S rats fed a high-salt diet (one-way ANOVA, F(2,19) time = 28.95, p&lt;0.0001), and ELP 63 -MMP-2i had no effect on the blood pressure elevation (F(2,9) treatment = 0.03, p=0.97). Proteinuria was also significantly and progressively elevated during the high-salt feeding period (F(2,19) time = 15.61, p&lt;0.0001). ELP 63 -MMP-2i treatment reduced the severity of proteinuria (F(2,9) treatment = 5.02, p=0.03). We also observed significant renal fibrosis in Dahl S rats after 21 days of high-salt diet, and that there was a trend for reduced fibrosis in the renal cortex in rats treated with ELP 63 -MMP-2i compared to vehicle (paired t-test, p=0.11). These are intermediate findings of an ongoing study with a planned enrollment of 8 rats per group. The intermediate analysis suggests that inhibiting MMP-2 activity with an MMP-2 specific and renally targeted biologic is renoprotective and may be beneficial as a treatment for CKD. This work was supported by NIH grants R01HL095638 to GLB, R01HL138685 to RJR and F31DK130598 to AAA. 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.

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.

TNF/iNOS/NO pathway mediates host susceptibility to endothelial-dependent circulatory failure and death induced by betacoronavirus infection

Poor disease outcomes and lethality are directly related to endothelial dysfunction in betacoronavirus infections. Here, we investigated the mechanisms underlying the vascular dysfunction caused by the betacoronaviruses MHV-3 and SARS-CoV-2. Wild-type C57BL/6 (WT) and knockout mice for inducible nitric oxide synthase (iNOS-/-) or TNF receptor 1 (TNFR1-/-) were infected with MHV-3, and K18-hACE2 transgenic mice expressing human ACE2 were infected with SARS-CoV-2. Isometric tension was used to evaluate vascular function. Protein expression was determined by immunofluorescence. Tail-cuff plethysmography and Doppler were used to assess blood pressure and flow, respectively. Nitric oxide (NO) was quantified with the DAF probe. ELISA was used to assess cytokine production. Survival curves were estimated using Kaplan-Meier. MHV-3 infection reduced aortic and vena cava contractility, arterial blood pressure, and blood flow, resulting in death. Resistance mesenteric arteries showed increased contractility. The contractility of the aorta was normalized by removing the endothelium, inhibiting iNOS, genetically deleting iNOS, or scavenging NO. In the aorta, iNOS and phospho-NF-kB p65 subunit expression was enhanced, along with basal NO production. TNF production was increased in plasma and vascular tissue. Genetic deletion of TNFR1 prevented vascular changes triggered by MHV-3, and death. Basal NO production and iNOS expression were also increased by SARS-CoV-2. In conclusion, betacoronavirus induces an endothelium-dependent decrease in contractility in macro-arteries and veins, leading to circulatory failure and death via TNF/iNOS/NO. These data highlight the key role of the vascular endothelium and TNF in the pathogenesis and lethality of coronaviruses.

Amlodipine limits microglia activation and cognitive dysfunction in aged hypertensive mice.

SBP and blood pressure variability are independent risk factors for cerebral small vessel disease, a leading cause for stroke and dementia. Calcium-channel blockers are known to reduce blood pressure variability and may thus offer benefit against dementia. Beyond this effect, the impact of calcium-channel blockers on hypertension-induced neuroinflammation, and especially, microglial phenotype remains unknown. We aimed to study the ability of amlopidine to alleviate microglia inflammation, and slow down cognitive dysfunction in aged hypertensive mice. Hypertensive BPH/2J and normotensive BPN/3J mice were studied until 12 months of age. Hypertensive mice were untreated or received amlodipine (10 mg/kg per day). Blood pressure parameters were measured by telemetry and tail cuff plethysmography. Mice underwent repeated series of cognitive tasks. Brain immunohistochemistry was performed to study blood-brain barrier dysfunction and microglial pro-inflammatory phenotype (CD68 + Iba1 + cells; morphological analysis). Amlodipine normalized SBP over the entire life span and decreased blood pressure variability. BPH/2J mice exhibited impaired short-term memory that was prevented by amlodipine at 12 months (discrimination index 0.41 ± 0.25 in amlodipine-treated vs. 0.14 ± 0.15 in untreated BPH/2J mice, P = 0.02). Amlopidine treatment of BPH/2J did not prevent blood-brain barrier leakage, a measure of cerebral small vessel disease, but limited its size. Microglia's inflammatory phenotype in BPH/2J, characterized by an increased number of Iba1 + CD68 + cells, increased soma size and shortened processes, was partly reduced by amlodipine. Amlodipine attenuated the short-term memory impairment in aged hypertensive mice. Beyond its blood pressure lowering capacity, amlodipine may be cerebroprotective by modulating neuroinflammation.