ß-arrestin biased signaling is not involved in the hypotensive actions of 5-HT7 receptor stimulation: use of Serodolin.

The 5-hydroxytryptamine 7 receptor (5-HT7) is necessary for 5-HT to cause a concentration-dependent vascular relaxation and hypotension. 5-HT7 is recognized as having biased signaling, transduced through either Gs or ß -arrestin. It is unknown whether 5-HT7 signals in a biased manner to cause vasorelaxation/hypotension. We used the recently described ß-arrestin selective 5-HT7 receptor agonist serodolin to test the hypothesis that 5-HT7 activation does not cause vascular relaxation or hypotension via the ß -arrestin pathway. Isolated abdominal aorta (no functional 5-HT7) and vena cava (functional 5-HT7) from male Sprague Dawley rats were used in isometric contractility studies. Serodolin (1 nM - 10 µM) did not change baseline tone of isolated tissues and did not relax the endothelin-1 (ET-1)-contracted vena cava or aorta. In the aorta, serodolin acted as a 5-HT2A receptor antagonist, evidenced by a rightward shift in 5-HT-induced concentration response curve [pEC50 5-HT [M]: Veh = 5.2 +/- 0.15; Ser (100 nM) = 4.49 +/- 0.08; p < 0.05]. In the vena cava, serodolin acted as a 5-HT7 receptor antagonist, shifting the concentration response curve to 5-HT left and upward (%10 µM NE contraction; Veh = 3.2 +/- 1.7; Ser (10 nM) = 58 +/- 11; p < 0.05) and blocking relaxation of pre-contracted tissue to the 5-HT1A/7 agonist 5-carboxamidotryptamine. In anesthetized rats, 5-HT or serodolin was infused at 5, 25 and 75 µg/kg/min, iv. Though 5-HT caused concentration-dependent depressor responses, serodolin caused an insignificant small depressor responses at all three infusion rates. With the final dose of serodolin on board, 5-HT was unable to reduce blood pressure. Collectively the data indicate that serodolin functions as a 5-HT7 antagonist with additional 5-HT2A blocking properties. 5-HT7 activation does not cause vascular relaxation or hypotension via the ß -arrestin pathway.

Chemerin is resident to vascular tunicas and contributes to vascular tone.

The adipokine chemerin may support blood pressure, evidenced by a fall in mean arterial pressure after whole body antisense oligonucleotide (ASO)-mediated knockdown of chemerin protein in rat models of normal and elevated blood pressure. Although the liver is the greatest contributor of circulating chemerin, liver-specific ASOs that abolished hepatic-derived chemerin did not change blood pressure. Thus, other sites must produce the chemerin that supports blood pressure. We hypothesize that the vasculature is a source of chemerin independent of the liver that supports arterial tone. RNAScope, PCR, Western blot analyses, ASOs, isometric contractility, and radiotelemetry were used in the Dahl salt-sensitive (SS) rat (male and female) on a normal diet. Retinoic acid receptor responder 2 (Rarres2) mRNA was detected in the smooth muscle, adventitia, and perivascular adipose tissue of the thoracic aorta. Chemerin protein was detected immunohistochemically in the endothelium, smooth muscle cells, adventitia, and perivascular adipose tissue. Chemerin colocalized with the vascular smooth muscle marker α-actin and the adipocyte marker perilipin. Importantly, chemerin protein in the thoracic aorta was not reduced when liver-derived chemerin was abolished by a liver-specific ASO against chemerin. Chemerin protein was similarly absent in arteries from a newly created global chemerin knockout in Dahl SS rats. Inhibition of the receptor Chemerin1 by the receptor antagonist CCX832 resulted in the loss of vascular tone that supports potential contributions of chemerin by both perivascular adipose tissue and the media. These data suggest that vessel-derived chemerin may support vascular tone locally through constitutive activation of Chemerin1. This posits chemerin as a potential therapeutic target in blood pressure regulation.NEW & NOTEWORTHY Vascular tunicas synthesizing chemerin is a new finding. Vascular chemerin is independent of hepatic-derived chemerin. Vasculature from both males and females have resident chemerin. Chemerin1 receptor activity supports vascular tone.

Male and female high-fat diet-fed Dahl SS rats are largely protected from vascular dysfunctions: PVAT contributions reveal sex differences.

Vascular dysfunctions are observed in the arteries from hypertensive subjects. The establishment of the Dahl salt-sensitive (SS) male and female rat models to develop a reproducible hypertension with high-fat (HF) diet feeding from weaning allows addressing the question of whether HF diet-associated hypertension results in vascular dysfunction similar to that of essential hypertension in both sexes. We hypothesized that dysfunction of three distinct vascular layers, i.e., endothelial, smooth muscle, and perivascular adipose tissue (PVAT), would be present in the aorta from HF diet-fed versus control diet-fed male and female rats. Dahl SS rats were fed a control (10% kcal of fat) or HF (60%) diet from weaning for 24 wk. Male and female Dahl SS rats became equally hypertensive when placed on a HF diet. For male and female rats, the thoracic aorta exhibited medial hypertrophy in HF diet-induced hypertension versus control, but neither displayed a hyperresponsive contraction to the α-adrenergic agonist phenylephrine nor an endothelial cell dysfunction as measured by acetylcholine-induced relaxation. A beneficial PVAT function, support of stress relaxation, was reduced in the male versus female rats fed a HF diet. PVAT in the aorta of males but not in females retained the anticontractile activity. We conclude that this HF model does not display the same vascular dysfunctions observed in essential hypertension. Moreover, both male and female show significantly different vascular dysfunctions in this HF feeding model.NEW & NOTEWORTHY Although the aorta exhibits medial hypertrophy in response to HF diet-induced hypertension, it did not exhibit hyperresponsive contraction to an α-adrenergic agonist nor endothelial cell dysfunction; this was true for both sexes. Unlike other hypertension models, PVAT around aorta from (male) rats on the HF diet retained significant anticontractile activity. PVAT around aorta of the male on a HF diet was modestly more fibrotic and lost the ability to assist in arterial stress relaxation.

Blood pressure changes PVAT function and transcriptome: use of the mid-thoracic aorta coarcted rat.

Perivascular adipose tissue (PVAT) modifies the contractile function of the vessel it surrounds (outside-in signaling). Little work points to the vessel actively affecting its surrounding PVAT. We hypothesized that inside-out arterial signaling to PVAT would be evidenced by the response of PVAT to changes in tangential vascular wall stress. Rats coarcted in the mid-thoracic aorta created PVAT tissues that would exemplify pressure-dependent changes (above vs. below coarctation); a sham rat was used as a control. Radiotelemetry revealed a ∼20 mmHg systolic pressure gradient across the coarctation 4 wk after surgery. Four measures (histochemical, adipocyte progenitor proliferation and differentiation, isometric tone, and bulk mRNA sequencing) were used to compare PVAT above versus below the ligature in sham and coarcted rats. Neither aortic collagen deposition in PVAT nor arterial media/radius ratio above coarctation was increased versus below segments. However, differentiated adipocytes derived from PVAT above the coarctation accumulated substantially less triglycerides versus those below; their relative proliferation rate as adipogenic precursors was not different. Functionally, the ability of PVAT to assist stress relaxation of isolated aorta was reduced in rings above versus below the coarctation. Transcriptomic analyses revealed that the coarctation resulted in more differentially expressed genes (DEGs) between PVAT above versus below when compared with sham samples from the same locations. A majority of DEGs were in PVAT below the coarctation and were enriched in neuronal/synaptic terms. These findings provide initial evidence that signaling from the vascular wall, as stimulated by a pressure change, influences the function and transcriptional profile of its PVAT.NEW & NOTEWORTHY A mid-thoracic aorta coarcted rat was created to generate a stable pressure difference above versus below the coarctation ligature. This study determined that the PVAT around the thoracic aorta exposed to a higher pressure has a significantly reduced ability to assist stress relaxation versus that below the ligature and appears to retain the ability to be anticontractile. At the same time, the PVAT around the thoracic aorta exposed to higher pressure had a reduced adipogenic potential versus that below the ligature. Transcriptomics analyses indicated that PVAT below the coarctation showed the greatest number of DEGs with an increased profile of the synaptic neurotransmitter gene network.

Different blood pressure responses in hypertensive rats following chemerin mRNA inhibition in dietary high fat compared to dietary high-salt conditions.

Chemerin is a contractile adipokine, produced in liver and fat, and removal of the protein by antisense oligonucleotides (ASO) lowers blood pressure in the normal Sprague Dawley rat. In humans, chemerin is positively associated with blood pressure and obesity so we hypothesized that in a model of hypertension derived from high-fat (HF) feeding, the chemerin ASO would reduce blood pressure more than a high-salt (HS) model. Male Dahl S rats were given a HF (60% kcal fat; age 3-24 wk) or HS diet (4% salt; age 20-24 wk to match age and blood pressure of HF animals). Scrambled control, whole body, or liver-specific ASOs that knock down chemerin were delivered subcutaneously once per week for 4 wk with tissue and blood collected 2 days after the last injection. Conscious blood pressure was measured 24 h/day by radiotelemetry. By the end of whole body ASO administration, blood pressure of HF animals had fallen 29 ± 2 mmHg below baseline, while blood pressure of HS-diet animals fell by only 12 ± 4 mmHg below baseline. Administration of a liver-specific ASO to HF Dahl S resulted in a 6 ± 2 mmHg fall in blood pressure below baseline. Successful knockdown of chemerin in both the whole body and liver-specific administration was confirmed by Western and PCR. These results suggest that chemerin, not derived from liver but potentially from adipose tissue, is an important driver of hypertension associated with high fat. This knowledge could lead to the development of antihypertensive treatments specifically targeted to obesity-associated hypertension.

The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension.

Measures of the adipokine chemerin are elevated in multiple cardiovascular diseases, including hypertension, but little mechanistic work has been done to implicate chemerin as being causative in such diseases. The chemerin knockout (KO) rat was created to test the hypothesis that removal of chemerin would reduce pressure in the normal and hypertensive state. Western analyses confirmed loss of chemerin in the plasma and tissues of the KO vs. wild-type (WT) rats. Chemerin concentration in plasma and tissues was lower in WT females than in WT males, as determined by Western analysis. Conscious male and female KO rats had modest differences in baseline measures vs. the WT that included systolic, diastolic, mean arterial and pulse pressures, and heart rate, all measured telemetrically. The mineralocorticoid deoxycorticosterone acetate (DOCA) and salt water, combined with uninephrectomy as a hypertensive stimulus, elevated mean and systolic blood pressures of the male KO higher than the male WT. By contrast, all pressures in the female KO were lower than their WT throughout DOCA-salt treatment. These results revealed an unexpected sex difference in chemerin expression and the ability of chemerin to modify blood pressure in response to a hypertensive challenge.-Watts, S. W., Darios, E. S., Mullick, A. E., Garver, H., Saunders, T. L., Hughes, E. D., Filipiak, W. E., Zeidler, M. G., McMullen, N., Sinal, C. J., Kumar, R. K., Ferland, D. J., Fink, G. D. The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension.

Macrophage-dependent impairment of α2-adrenergic autoreceptor inhibition of Ca2+ channels in sympathetic neurons from DOCA-salt but not high-fat diet-induced hypertensive rats.

DOCA-salt and obesity-related hypertension are associated with inflammation and sympathetic nervous system hyperactivity. Prejunctional α2-adrenergic receptors (α2ARs) provide negative feedback to norepinephrine release from sympathetic nerves through inhibition of N-type Ca2+ channels. Increased neuronal norepinephrine release in DOCA-salt and obesity-related hypertension occurs through impaired α2AR signaling; however, the mechanisms involved are unclear. Mesenteric arteries are resistance arteries that receive sympathetic innervation from the superior mesenteric and celiac ganglia (SMCG). We tested the hypothesis that macrophages impair α2AR-mediated inhibition of Ca2+ channels in SMCG neurons from DOCA-salt and high-fat diet (HFD)-induced hypertensive rats. Whole cell patch-clamp methods were used to record Ca2+ currents from SMCG neurons maintained in primary culture. We found that DOCA-salt, but not HFD-induced, hypertension caused macrophage accumulation in mesenteric arteries, increased SMCG mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-α, and impaired α2AR-mediated inhibition of Ca2+ currents in SMCG neurons. α2AR dysfunction did not involve changes in α2AR expression, desensitization, or downstream signaling factors. Oxidative stress impaired α2AR-mediated inhibition of Ca2+ currents in SMCG neurons and resulted in receptor internalization in human embryonic kidney-293T cells. Systemic clodronate-induced macrophage depletion preserved α2AR function and lowered blood pressure in DOCA-salt rats. HFD caused hypertension without obesity in Sprague-Dawley rats and hypertension with obesity in Dahl salt-sensitive rats. HFD-induced hypertension was not associated with inflammation in SMCG and mesenteric arteries or α2AR dysfunction in SMCG neurons. These results suggest that macrophage-mediated α2AR dysfunction in the mesenteric circulation may only be relevant to mineralocorticoid-salt excess. NEW & NOTEWORTHY Here, we identify a contribution of macrophages to hypertension development through impaired α2-adrenergic receptor (α2AR)-mediated inhibition of sympathetic nerve terminal Ca2+ channels in DOCA-salt hypertensive rats. Impaired α2AR function may involve oxidative stress-induced receptor internalization. α2AR dysfunction may be unique to mineralocorticoid-salt excess, as it does not occur in obesity-related hypertension.

Aging is associated with changes to the biomechanical properties of the posterior cerebral artery and parenchymal arterioles.

Artery remodeling, described as a change in artery structure, may be responsible for the increased risk of cardiovascular disease with aging. Although the risk for stroke is known to increase with age, relatively young animals have been used in most stroke studies. Therefore, more information is needed on how aging alters the biomechanical properties of cerebral arteries. Posterior cerebral arteries (PCAs) and parenchymal arterioles (PAs) are important in controlling brain perfusion. We hypothesized that aged (22-24 mo old) C57bl/6 mice would have stiffer PCAs and PAs than young (3-5 mo old) mice. The biomechanical properties of the PCAs and PAs were assessed by pressure myography. Data are presented as means ± SE of young vs. old. In the PCA, older mice had increased outer (155.6 ± 3.2 vs. 169.9 ± 3.2 µm) and lumen (116.4 ± 3.6 vs. 137.1 ± 4.7 µm) diameters. Wall stress (375.6 ± 35.4 vs. 504.7 ± 60.0 dyn/cm(2)) and artery stiffness (ß-coefficient: 5.2 ± 0.3 vs. 7.6 ± 0.9) were also increased. However, wall strain (0.8 ± 0.1 vs. 0.6 ± 0.1) was reduced with age. In the PAs from old mice, wall thickness (3.9 ± 0.3 vs. 5.1 ± 0.2 µm) and area (591.1 ± 95.4 vs. 852.8 ± 100 µm(2)) were increased while stress (758.1 ± 100.0 vs. 587.2 ± 35.1 dyn/cm(2)) was reduced. Aging also increased mean arterial and pulse pressures. We conclude that age-associated remodeling occurs in large cerebral arteries and arterioles and may increase the risk of cerebrovascular disease.

DOCA-salt hypertension impairs artery function in rat middle cerebral artery and parenchymal arterioles.

OBJECTIVE: Chronic hypertension induces detrimental changes in the structure and function of surface cerebral arteries. Very little is known about PAs, which perfuse distinct neuronal populations in the cortex and may play a role in cerebrovascular disorders. We investigated the effect of DOCA-salt induced hypertension on endothelial function and artery structure in PAs and MCAs. METHODS: Uninephrectomized male Sprague-Dawley rats were implanted with a subcutaneous pellet containing DOCA (150 mg/kg b.w.) and drank salt water (1% NaCl and 0.2% KCl) for 4 weeks. Sham rats were uninephrectomized and drank tap water. Vasoreactivity and passive structure in the MCAs and the PAs were assessed by pressure myography. RESULTS: Both MCAs and PAs from DOCA-salt rats exhibited impaired endothelium-dependent dilation (P<.05). In the PAs, addition of NO and COX inhibitors enhanced dilation in DOCA-salt rats (P<.05), suggesting that dysfunctional NO and COX-dependent signaling could contribute to impaired endothelium-mediated dilation. MCAs from DOCA-salt rats exhibited inward remodeling (P<.05). CONCLUSIONS: Hypertension-induced MCA remodeling coupled with impaired endothelium-dependent dilation in both the MCAs and PAs may exacerbate the risk of cerebrovascular accidents and the associated morbidity and mortality.

Altered L-type Ca2+ channel activity contributes to exacerbated hypoperfusion and mortality in smooth muscle cell BK channel-deficient septic mice.

We determined the contribution of vascular large conductance Ca2+-activated K+ (BK) and L-type Ca2+ channel dysregulation to exaggerated mortality in cecal ligation/puncture (CLP)-induced septic BK channel ß1-subunit knockout (BK ß1-KO, smooth muscle specific) mice. CLP-induced hemodynamic changes and mortality were assessed over 7 days in wild-type (WT) and BK ß1-KO mice that were either untreated, given volume resuscitation (saline), or saline + calcium channel blocker nicardipine. Some mice were euthanized 24 h post-CLP to measure tissue injury and vascular and immune responses. CLP-induced hypotension was similar in untreated WT and BK ß1-KO mice, but BK ß1-KO mice died sooner. At 24 h post-CLP (mortality latency in BK ß1-KO mice), untreated CLP-BK ß1-KO mice showed more severe hypothermia, lower tissue perfusion, polymorphonuclear neutrophil infiltration-independent severe intestinal necrosis, and higher serum cytokine levels than CLP-WT mice. Saline resuscitation improved survival in CLP-WT but not CLP-BK ß1-KO mice. Saline + nicardipine-treated CLP-BK ß1-KO mice exhibited longer survival times, higher tissue perfusion, less intestinal injury, and lower cytokines versus untreated CLP-BK ß1-KO mice. These improvements were absent in treated CLP-WT mice, although saline + nicardipine improved blood pressure similarly in both septic mice. At 24 h post-CLP, BK and L-type Ca2+ channel functions in vitro were maintained in mesenteric arteries from WT mice. Mesenteric arteries from BK ß1-KO mice had blunted BK/enhanced L-type Ca2+ channel function. We conclude that vascular BK channel deficiency exaggerates mortality in septic BK ß1-KO mice by activating L-type Ca2+ channels leading to blood pressure-independent tissue ischemia.

Skin Vascular Resistance Decreases during 5-HT-Induced Hypotension in the Rat.

A recognized vasodilator, the infusion of 5-hydroxytryptamine (5-HT, serotonin) decreases blood pressure through the reduction of total peripheral resistance in the rat. It is not clear which vascular beds/tissues are responsible for this fall. We hypothesized that an increase in blood flow within the skin, measured as an elevated temperature (T) in the thermoregulatory tail and paws, enables at least part of 5-HT-induced reduction in blood pressure through active vasodilation. The temperature of thermoregulatory regions of the skin of an anesthetized male, Sprague Dawley rats were measured using a Optris PI640 thermal camera. The blood pressure of the animal and the temperature of each paw and four locations along the tail (TL1-4) were recorded before, during, and after the infusion of 5-HT at a rate of 25 mg/min into a femoral vein. Contrary to our hypothesis, the temperature of the paws and tail was stable before and during 5-HT infusion and actually increased during the 15-min recovery period. This finding suggests that hyperemia of the skin circulation is not necessary for the fall in blood pressure observed with infused 5-HT, but that a reduction in cutaneous vascular resistance plays a part in the fall in total peripheral resistance.

Vascular BK channel deficiency exacerbates organ damage and mortality in endotoxemic mice.

We determined the contribution of vascular BK channels to endotoxin (lipopolysaccharide, LPS)-induced hypotension, organ damage, and mortality using smooth muscle BK channel deficiency (BK channel ß1-subunit knockout, BK ß1-KO) mice. BK ß1-KO mice were more sensitive to LPS-induced mortality compared with wild-type mice. After LPS (20 mg/kg, intraperitoneally), BK ß1-KO mice had a more rapid fall in heart rate and blood pressure (measured by radiotelemetry), shorter latency to mortality, and higher mortality rate than wild-type mice. Twenty-two hours after LPS treatment, wild-type and BK ß1-KO mice had reduced norepinephrine reactivity and impaired constrictor responses to the BK channel blocker paxilline in mesenteric arteries in vitro and higher iNOS expression in the heart, but not in mesenteric arteries. Endotoxemic BK ß1-KO mice also showed more severe lung and intestinal injury, higher myeloperoxidase activity and polymorphonuclear neutrophil infiltration in lung and liver. Endotoxemic BK ß1-KO mice had higher plasma tumor necrosis factor α and interleukin 6 levels at 22 hours, but not 6 hours post-LPS. Exaggerated mortality in BK ß1-KO mice also occurred in the cecal ligation/puncture model of septic shock. Reduced vascular BK channel function does not protect against hypotension in the early stage of septic shock; in the later stage, smooth muscle BK channel deficiency enhances organ damage and mortality.

A high fat diet does not stimulate blood pressure dependence on chemerin in the Sprague-Dawley rat.

The adipokine chemerin is a candidate for connecting obesity to hypertension. Study objective: To test the hypothesis that a high fat (HF) diet stimulates dependence on chemerin for blood pressure regulation. Design: Blood pressure in male Sprague Dawley rats fed a control (10 % fat) or HF (60 % fat) diet from weaning was measured using radiotelemetry. Antisense oligonucleotides (ASOs), administered after 17 weeks of feeding, were used to abolish chemerin production. Results: The HF diet did not increase blood pressure (mm Hg; control = 117.0 ± 2.5; HF = 122.0 ± 2.2). An ASO against chemerin (dosed 1×/week, 4 weeks) similarly reduced blood pressure in the control (-14.0 ± 2.7 mmHg) and HF rat (-12.4 ± 2.3). Chemerin mRNA was abolished in the liver and fats (primary producers of chemerin) from rats given the ASO chemerin vs control. Conclusion: A HF diet alone is insufficient to stimulate the dependence of blood pressure in the rat on chemerin.

Large-conductance Ca2+-activated K+ channel beta1-subunit knockout mice are not hypertensive.

Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory ß1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel ß1-subunit knockout (BK ß1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK ß1-KO mice using radiotelemetry. BK ß1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ∼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK ß1-KO mice. In BK ß1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (∼5 mmHg); MAP returned to pre-saline levels by day 6. BK ß1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK ß1-KO mesenteric veins (MV). BK ß1-subunits are not expressed in MV. The results indicate that BK ß1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK ß1-KO mice. BK and L-type Ca(2+) channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension.

Doxycycline, a matrix metalloprotease inhibitor, reduces vascular remodeling and damage after cerebral ischemia in stroke-prone spontaneously hypertensive rats.

Matrix metalloproteases (MMPs) are a family of zinc peptidases involved in extracellular matrix turnover. There is evidence that increased MMP activity is involved in remodeling of resistance vessels in chronic hypertension. Thus we hypothesized that inhibition of MMP activity with doxycycline (DOX) would attenuate vascular remodeling. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with DOX (50 mg·kg(-1)·day(-1) in the drinking water) for 6 wk. Untreated SHRSP were controls. Blood pressure was measured by telemetry during the last week. Middle cerebral artery (MCA) and mesenteric resistance artery (MRA) passive structures were assessed by pressure myography. MMP-2 expression in aortas was measured by Western blot. All results are means ± SE. DOX caused a small increase in mean arterial pressure (SHRSP, 154 ± 1; SHRSP + DOX, 159 ± 3 mmHg; P < 0.001). Active MMP-2 expression was reduced in aorta from SHRSP + DOX (0.21 ± 0.06 vs. 0.49 ± 0.13 arbitrary units; P < 0.05). In the MCA, at 80 mmHg, DOX treatment increased the lumen (273.2 ± 4.7 vs. 238.3 ± 6.3 µm; P < 0.05) and the outer diameter (321 ± 5.3 vs. 290 ± 7.6 µm; P < 0.05) and reduced the wall-to-lumen ratio (0.09 ± 0.002 vs. 0.11 ± 0.003; P < 0.05). Damage after transient cerebral ischemia (transient MCA occlusion) was reduced in SHRSP + DOX (20.7 ± 4 vs. 45.5 ± 5% of hemisphere infarcted; P < 0.05). In the MRA, at 90 mmHg DOX, reduced wall thickness (29 ± 1 vs. 22 ± 1 µm; P < 0.001) and wall-to-lumen ratio (0.08 ± 0.004 vs. 0.11 ± 0.008; P < 0.05) without changing lumen diameter. These results suggest that MMPs are involved in hypertensive vascular remodeling in both the peripheral and cerebral vasculature and that DOX reduced brain damage after cerebral ischemia.

Chronic estradiol-17ß exposure increases superoxide production in the rostral ventrolateral medulla and causes hypertension: reversal by resveratrol.

Women are exposed to estrogen in several forms, such as oral contraceptive pills and hormone replacement therapy. Although estrogen was believed to be cardioprotective, lately, its beneficial effects are being questioned. Recent studies indicate that oxidative stress in the rostral ventrolateral medulla (RVLM) may play a role in the development of hypertension. Therefore, we hypothesized that chronic exposure to low levels of estradiol-17ß (E(2)) leads to hypertension in adult-cycling female Sprague Dawley (SD) rats potentially through generation of superoxide in the RVLM. To test this hypothesis, young adult (3 or 4 mo old) female SD rats were either sham-implanted or implanted (subcutaneously) with slow-release E(2) pellets (20 ng/day) for 90 days. A group of control and E(2)-treated animals were fed lab chow or chow containing resveratrol (0.84 g/kg of chow), an antioxidant. Rats were implanted with telemeters to continuously monitor blood pressure (BP) and heart rate (HR). At the end of treatment, the RVLM was isolated for measurements of superoxide. E(2) treatment significantly increased mean arterial pressure (mmHg) and HR (beats/min) compared with sham rats (119.6 ± 0.8 vs. 105.1 ± 0.7 mmHg and 371.7 ± 1.5 vs. 354.4 ± 1.3 beats/min, respectively; P < 0.0001). Diastolic and systolic BP were significantly increased in E(2)-treated rats compared with control animals. Superoxide levels in the RVLM increased significantly in the E(2)-treated group (0.833 ± 0.11 nmol/min·mg) compared with control (0.532 ± 0.04 nmol/min·mg; P < 0.05). Treatment with resveratrol reversed the E(2)-induced increases in BP and superoxide levels in the RVLM. In conclusion, these findings support the hypothesis that chronic exposure to low levels of E(2) induces hypertension and increases superoxide levels in the RVLM and that this effect can be reversed by resveratrol treatment.

Renal Denervation and Celiac Ganglionectomy Decrease Mean Arterial Pressure Similarly in Genetically Hypertensive Schlager (BPH/2J) Mice.

Renal denervation (RDNX) lowers mean arterial pressure (MAP) in patients with resistant hypertension. Less well studied is the effect of celiac ganglionectomy (CGX), a procedure which involves the removal of the nerves innervating the splanchnic vascular bed. We hypothesized that RDNX and CGX would both lower MAP in genetically hypertensive Schlager (BPH/2J) mice through a reduction in sympathetic tone. Telemeters were implanted into the femoral artery in mice to monitor MAP before and after RDNX (n=5), CGX (n=6), or SHAM (n=6). MAP, systolic blood pressure, diastolic blood pressure, and heart rate were recorded for 14 days postoperatively. The MAP response to hexamethonium (10 mg/kg, IP) was measured on control day 3 and postoperative day 10 as a measure of global neurogenic pressor activity. The efficacy of denervation was assessed by measurement of tissue norepinephrine. Control MAP was similar among the 3 groups before surgical treatments (≈130 mm Hg). On postoperative day 14, MAP was significantly lower in RDNX (-11±2 mm Hg) and CGX (-11±1 mm Hg) groups compared with their predenervation values. This was not the case in SHAM mice (-5±3 mm Hg). The depressor response to hexamethonium in the RDNX group was significantly smaller on postoperative day 10 (-10±5 mm Hg) compared with baseline control (-25±10 mm Hg). This was not the case in mice in the SHAM (day 10; -28±5 mm Hg) or CGX (day 10; -34±7 mm Hg) group. In conclusion, both renal and splanchnic nerves contribute to hypertension in BPH/2J mice, but likely through different mechanisms.

Phenotypic Changes in T Cell and Macrophage Subtypes in Perivascular Adipose Tissues Precede High-Fat Diet-Induced Hypertension.

Perivascular adipose tissue (PVAT) may connect adiposity to hypertension because of its vasoactive functions and proximity to blood vessels. We hypothesized that immune cell changes in PVATs precede the development of high fat diet (HFD)-induced hypertension. Both sexes of Dahl S rat become equally hypertensive when fed a HFD. Further, both sexes would have similar immune cell composition in PVATs with the development and progression of hypertension. Male and female Dahl S rats were fed a regular (10% calories from fat; CD) diet or a HFD (60%) from weaning. PVATs from around the thoracic aorta (APVAT) and small mesenteric vessels (MRPVAT) were harvested at 10 weeks (pre-hypertensive), 17 weeks (onset), or 24 (hypertensive) weeks on diet. RNA-sequencing in MRPVAT at 24 weeks indicated sex-differences with HFD (>CD) and diet-differences in males (>females). The top 2 out of 7 immune processes with the maximum number of differentially expressed genes (DEGs) were associated with immune effector processes and leukocyte activation. Macrophages and T cells (and their activation status), neutrophils, mast, B and NK cells were measured by flow cytometry. Sex-specific changes in the number of CD4 memory T cells (males > females) and M2-like macrophages (females > males) in PVATs occur with a HFD before hypertension developed. Sex-differences became more prominent with the development and progression of hypertension, driven by the diet (HFD > CD). These findings suggest that though the magnitudes of increased blood pressure were equivalent in both sexes, the associated phenotypic changes in the immune subsets within the PVATs were different in the male vs. the female with the development and progression of hypertension.

Activation of ventral tegmental area neurotensin Receptor-1 neurons promotes weight loss.

Dopamine (DA) neurons in the ventral tegmental area (VTA) modulate physical activity and feeding behaviors that are disrupted in obesity. Yet, the heterogeneity of VTA DA neurons has hindered determination of which ones might be leveraged to support weight loss. We hypothesized that increased activity in the subset of VTA DA neurons expressing neurotensin receptor-1 (NtsR1) might promote weight loss behaviors. To test this, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to activate VTA NtsR1 neurons in normal weight and diet-induced obese mice. Acute activation of VTA NtsR1 neurons (24hr) significantly decreased body weight in normal weight and obese mice by reducing food intake and increasing physical activity. Moreover, daily activation of VTA NtsR1 neurons in obese mice sustained weight loss over 7 days. Activating VTA NtsR1 neurons also suppressed how much mice worked to obtain sucrose rewards, even when there was high motivation to consume. However, VTA NtsR1 neural activation was not reinforcing, nor did it invoke liabilities associated with whole-body NtsR1 agonism such as anxiety, vasodepressor response or hypothermia. Activating VTA NtsR1 neurons therefore promotes dual behaviors that support weight loss without causing adverse effects, and is worth further exploration for managing obesity.

Chemerin contributes to in vivo adipogenesis in a location-specific manner.

Since chemerin's identification as an adipokine, it has been associated with a number of human diseases including diabetes and obesity. However, the basic scientific foundation for these clinical determinations is still lacking. Fibroblastic mouse 3T3 cells are unable to develop lipid droplets if chemerin is not present. Thus, we hypothesized that an in vivo rat model chemerin knockout (KO; an advancement from the previously mentioned in vitro cultures) would have limited accumulation of lipid in adipocytes compared to their wild-type (WT) counterparts. Female WT/KO rats (Sprague Dawley background) were fed a low-fat diet starting at 8 weeks of age with weekly body weight and food consumption monitoring. At 25 weeks of age, adipose tissue depots were dissected and flash frozen for PCR analysis or fixed with paraformaldehyde for histology. Over the 17 weeks of experimentation, WT and KO animals did not have differences in total body weight or food consumption but KO animals had a significantly reduced amount of visceral fat compared to WT animals (via microCT at 8 and 25 weeks). Histology of retroperitoneal and mesenteric depots demonstrated a significant leftward shift in adipocyte size in the mesenteric but not the retroperitoneal depot of the KO compared to WT animals. Similarly, in the mesenteric fat of the KO rat, gene expression of adiponectin, fatty acid synthase, perilipin, and leptin were significantly reduced compared to mesenteric fat of WT animals and retroperitoneal fat of both WT and KO animals. Adiponectin was highlighted by a protein-protein interaction network as being important for the physiological effects of chemerin removal. These data are the first, to our knowledge, to demonstrate chemerin's adipokine potential in vivo and identify it as fat depot location-specific.