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Vascular remodeling is a feature of small vessel disease (SVD), predictive of vascular events, brain injury, and cognitive decline. Hypertension alters vascular structure including diameter and cross-sectional area (CSA) of the vessel wall. While effects of hypertension on large arteries have been studied relatively widely, detailed quantification, including recovery from hypertension in microvessels, is rare. Previously, angiotensin II (Ang II)-dependent hypertension produced increased CSA but reduced internal diameter (ID) of cerebral arterioles (inward remodeling) in brain. Because cerebral arterioles are targets of SVD, we examined the hypothesis that brain microvessels can recover from hypertension after return of mean arterial pressure (MAP) to normal levels. MAP was measured using radiotelemetry in adult male C57BL6J mice at baseline, during infusion of vehicle or Ang II (1.4 mg/kg per day using osmotic mini-pumps) for 28 days, and during a 28 day recovery period (n=5 in each group). Prior to treatment, MAP was similar in both groups, with MAP remaining stable in vehicle treated mice. With Ang II treatment, MAP began to rise on day 3, steadily increasing until day 28. On day 30, MAP began to decrease, reaching levels seen with vehicle on days 46-47. In anesthetized mice, we measured pressure, diameter, and CSA of the vessel wall in maximally dilated arterioles (baseline diameter of 62±3 microns) at 1, 3, 7, 14, 28, and 56 days after pump implantation (n=7-9 in each group). At day 1, ID and CSA were similar in both groups. With vehicle, there was no significant change in CSA or ID at any time point. With Ang II, CSA increased at day 7 and was maintained at days 14 and 28 (P=0.023). ID did not change at day 3 or 7, but was reduced (by ~15%, P=0.011) at 14 and 28 days. During recovery (day 56), CSA returned 63% of the way to normal (compared to vehicle), while ID remained at day 14 and 28 values. In conclusion, CSA changed rapidly during hypertension onset and largely recovered with a reduction in MAP. Inward remodeling developed slowly and did not recover after return of MAP to control levels. The lack of recovery after hypertension has implications for the impact of SVD including hypoperfusion, impaired vasodilation, and augmented injury during ischemia.

Hypertension alters vascular structure including diameter, cross-sectional area (CSA), and stiffness of the vessel wall. Inward remodeling is a feature of small vessel disease (SVD), predictive of vascular events and cognitive decline. While effects of prevention of hypertension on large arteries have been studied widely, quantification of temporal changes and recovery from hypertension in microvessels is rare. Previously, angiotensin II (Ang II)-dependent hypertension produced increased CSA but reduced internal diameter (ID) of maximally dilated arterioles (inward remodeling) in brain. Because cerebral arterioles are targets of SVD, we examined time course and whether microvessels recover from hypertension. Mean arterial pressure (MAP) was measured using radiotelemetry in adult male C57BL6J mice prior to, during infusion of vehicle or Ang II (1.4 mg/kg per day using osmotic mini-pumps) for 28 days, and a 28 day recovery period. Prior to treatment, MAP was similar in both groups. Throughout recording, MAP was stable in vehicle treated mice. With Ang II, MAP began to rise on day 3, steadily increasing until day 28. On day 30, MAP began to decrease, reaching levels seen with vehicle on days 46-47. In anesthetized mice, we measured pressure, diameter, and CSA of the vessel wall (histologically) in maximally dilated arterioles (baseline diameter of 63±1 microns) at 1, 3, 7, 14, 28, and 56 days after pump implantation (n=7-9 in each group). At day 1, ID and CSA were similar in both groups. With vehicle, there was no significant change in CSA or ID at any time point. With Ang II, CSA increased at day 7 and was maintained at days 14 and 28 (P=0.023). ID did not change at day 3 or 7, but was reduced (by ~15%, P=0.011) at 14 and 28 days. During recovery (day 56). CSA returned 63% of the way to normal (compared to vehicle), while ID remained at day 14 and 28 values. In conclusion, CSA changed rapidly during hypertension onset and largely recovered with a reduction in MAP. Inward remodeling developed slowly and did not return to vehicle levels with MAP. The lack of recovery of ID after a period of hypertension has implications for the impact of SVD including hypoperfusion, impaired vasodilation, and augmented injury during ischemia.

Background Vasovagal reflex is the most common form of syncope, but the pathophysiological mechanisms that initiate the reflex are not well understood. We aimed to study supine and early orthostatic levels of the neurohormones involved in control of circulatory homeostasis in relation to the onset of tilt‐induced vasovagal syncope (VVS). Methods and Results A total of 827 patients who were investigated for unexplained syncope with head‐up tilt test (HUT) and optional nitroglycerin provocation (Italian protocol) had blood samples collected while supine and after 3‐minutes of HUT. Of these, 173 (20.9%) patients developed VVS during drug‐free HUT, 161 of whom (males 44.7%; age 45±21 years) had complete data. We analyzed levels of epinephrine, norepinephrine, C‐terminal pro–arginine vasopressin, C‐terminal endothelin‐1, and midregional fragments of pro–atrial natriuretic peptide and pro‐adrenomedullin in relation to time from tilt‐up to onset of VVS. We applied a linear regression model adjusted for age and sex. The mean time to syncope was 11±7 minutes. Older age (β=0.13; SE=0.03, P, See Editorial Williford et al

Rationale: Renal inflammation contributes to the pathophysiology of hypertension. CD161a + immune cells are dominant in the (SHR) spontaneously hypertensive rat and expand in response to nicotinic cholinergic activation. Objective: We aimed to phenotype CD161a + immune cells in prehypertensive SHR after cholinergic activation with nicotine and determine if these cells are involved in renal inflammation and the development of hypertension. Methods and Results: Studies used young SHR and WKY (Wistar–Kyoto) rats. Splenocytes and bone marrow cells were exposed to nicotine ex vivo, and nicotine was infused in vivo. Blood pressures, kidney, serum, and urine were obtained. Flow cytometry, Luminex/ELISA, immunohistochemistry, confocal microscopy, and Western blot were used. Nicotinic cholinergic activation induced proliferation of CD161a + /CD68 + macrophages in SHR-derived splenocytes, their renal infiltration, and premature hypertension in SHR. These changes were associated with increased renal expression of MCP-1 (monocyte chemoattractant protein-1) and VLA-4 (very-late antigen-4). LLT1 (lectin-like transcript 1), the ligand for CD161a, was overexpressed in SHR kidney, whereas vascular cellular and intracellular adhesion molecules were similar to those in WKY. Inflammatory cytokines were elevated in SHR kidney and urine after nicotine infusion. Nicotine-mediated renal macrophage infiltration/inflammation was enhanced in denervated kidneys, not explained by angiotensin II levels or expression of angiotensin type-1/2 receptors. Moreover, expression of the anti-inflammatory α7-nAChR (α7-nicotinic acetylcholine receptor) was similar in young SHR and WKY rats. Conclusions: A novel, inherited nicotinic cholinergic inflammatory effect exists in young SHR, measured by expansion of CD161a + /CD68 + macrophages. This leads to renal inflammation and premature hypertension, which may be partially explained by increased renal expression of LLT-1, MCP-1, and VLA-4.

High fat diet (HFD) leads to obesity and the metabolic syndrome. However, the effect are less pronounced in females than in males. We suggest that a gender difference is in their response to the satiety peptide Cholecystokinin (CCK). We previously reported that the Ca 2+ -activated Cl - channel (CaCC) subunit Ano2 /TMEM16B is essential for the CCK-induced current in intestinal nodose neurons in male mice, and is down regulated in obese mice fed on HFD. We hypothesize that the CCK-induced current in female mice is mediated predominantly by Ano1 /TMEM16A, the alternative subunit of TMEM16B. We studied the CCK-induced current in nodose neurons from female C57BL/6 mice using whole cell patch-clamp. We found that 10nM of CCK induces an inward current in these neurons with a current density averaging 13.4±2.5 pA/pF (n=7), and this current is inhibited to 3.1±1.2 pA/pF (n=7, pinhi-A01 . We further tested whether HFD which effectively suppressed the expression of TMEM16B in male mice, would also decrease TMEM16A in female mice. Our results show that the CCK-induced TMEM16A current in nodose neurons is not decreased in female, but tend to increase from 12.5±1.6 (n=10) in control to 17.4±4.1 pA/pF (n=11, p=0.298) in female mice fed on HFD. Concurrently the mRNA level of Ano1 in mice fed on HFD also show a trend to increase by 1.33±0.41 fold (n=2 pairs of mice, p=0.37) compared to mice on regular chow. Our findings indicate that activation of CCK-sensitive Ano1 /TMEM16A may partially protect female mice from HFD induced obesity and metabolic syndrome.

Sarcomere gene mutations cause hypertrophic cardiomyopathy (HCM). Hallmark features include cardiac hypertrophy, fibrosis, excessive cardiac sympathetic tone, and increased risk of arrhythmias and sudden death. The mechanism(s) of increased sympathetic tone is poorly understood. We hypothesized that cardiac spinal (sympathetic) afferents are sensitized by metabolites (e.g., H+) produced during myocyte energy depletion in HCM, resulting in an enhanced cardiac sympathetic afferent reflex (CSAR). We studied mice with cardiac-specific expression of mutated alpha-tropomyosin (Glu180Gly), an established model of HCM. The CSAR was assessed by measuring mean arterial pressure (MAP) and heart rate (HR) responses to epicardial application of the TRPV1 agonist capsaicin (2.5mM, 2μL) to the left ventricle in anesthetized, ventilated HCM (n=5) and wild-type (WT) littermate control (n=5) mice. Pressor and tachycardic responses to capsaicin were markedly enhanced in HCM mice (Table). Blocking the tonic influence of cardiac afferent activity by epicardial application of the local anesthetic lidocaine (2%) caused small, significant decreases in MAP in both groups of mice; but decreased HR markedly in HCM mice only (n=5) (Table). mRNA expression (qPCR) of acid-sensing ion channel 3 was increased by 2-fold in dorsal root ganglia (T1-T9) of HCM vs. WT mice (n=6 each, P

Cholecystokinin (CCK) is a well-known satiety peptide that inhibits food intake. The CCK induced satiety signal in intestinal vagal afferents is attenuated in mice on a High Fat Diet (HFD). We have shown that down regulation of a Ca 2+ -activated Cl - channel (CaCC) downstream of CCK receptors is responsible for the neuronal insensitivity to CCK. We also found that the CaCC subunit Ano2/TMEM16B is essential for the CCK-induced current in nodose neurons. In this study we tested the hypothesis that reduction of this subunit in vivo contributes to weight gain. One allele of the Ano2/TMEM16B was knocked out in sensory neurons by crossing the ano2 fl/ fl mice with Nav1.8Cre mice to generate the Nav1.8Cre;ano2 fl /wt mice. The Cre negative littermates were used as control. We found that CCK-induced suppression of food intake is eliminated in male Nav1.8Cre;ano2 fl /wt mice. Food intake measured over 4 hours was 1.21±0.11g (n=5) in male wild type (wt) mice injected with saline and was reduced to 0.77±0.18 g (n=7, p0.05) in CCK injected male Nav1.8Cre;ano2 fl /wt mice. However, the CCK injection did not affect food intake in either female wt or Nav1.8Cre;ano2 fl /wt mice. The male Nav1.8/ano2 fl/wt mice were on the average 5.5g heavier than wt mice at 40 weeks of age (39.8±1.4 g, n=13 vs 34.3±1.4 g, n=14, pfl /wt mice (30.4±1.0 g, n=14 vs 28.5±1.0 g, n=11, p=0.20 respectively). Single cell mRNA level of Ano2 and CCK-induced TMEM16 currents in nodose neurons were reduced significantly in male Nav1.8Cre;ano2 fl/wt mice compared to Cre negative controls, but such changes were not seen in female mice. We conclude that heterozygote knockout of Ano2/TMEM16B specifically in sensory neurons causes neuronal insensitivity to CCK and excessive weight gain in male but not female mice. Reduction of this subunit may contribute to the HFD induced obesity. The reason for the phenotype and allele expression variability between sexes is unclear.

In vivo cholinergic activation with nicotine induces renal infiltration of M1 inflammatory CD161a+/CD68+ macrophages and the development of hypertension. Renal denervation has been proposed as a treatment for essential hypertension. Based on this we hypothesized that the renal sympathetic nerves play a role in these processes. Bilateral renal denervation was performed surgically in 3-4 week old Spontaneously Hypertensive Rats (SHR), a genetic model of essential hypertension. Following one week recovery, animals received subcutaneous infusion of nicotine (15mg/kg/day) via osmotic pumps for 2 weeks. Blood pressure was measured by tail-cuff and kidneys harvested on completion of infusion. Prior to nicotine infusion, at baseline, there was no difference between the systolic blood pressures of the sham treated (n=7) and renal denervation (n=10) groups, 132 ± 4 vs 128 ± 3 mmHg, respectively (p>0.05). In contrast, nicotine infusion significantly raised the systolic blood pressure in the sham treated group (159 ± 3mmHg), but not in the renal denervation group (135 ± 5 mmHg) (p

The innate immune system plays a key role in onset and maintenance of hypertension. We showed that the toll-like receptor (TLR) adaptor protein TRIF (toll interleukin receptor domain-containing adaptor-inducing interferon-b), but not MyD88 (myeloid differentiation 88), is required for angiotensin II (Ang II) induced hypertension in mice. TRIF transduces signals from TLR3 and TLR4, to induce inflammatory gene expression. In this study, we sought to determine which TLR is responsible for TRIF-mediated hypertensive effects of Ang II. We used a TLR4 deficient mice (TLR4-del) that have TLR4 gene deleted and the wild type (WT) control strain (C57BL/10). We also used a TLR3-knockout mice (TLR3ko) that have the exon 1 of the Tlr3 gene deleted. Saline or Ang II (1000 ng/kg/min) was infused subcutaneously for 3 weeks using mini-osmotic pumps and systolic blood pressure (SBP) was measured by tail cuff. Ang II increased peak SBP in WT (from 108 ± 1.3 mmHg to 136.0 ± 12.7 mmHg, n= 3) and TLR4-del (from 115.6 ± 2.7 mmHg to 154.4 ± 3.3 mmHg, n=3) mice. In contrast, peak SBP was not increased significantly with Ang II infusion in TLR3ko mice (from 111.3 ± 4.5 to 122.2 ± 18.6 mmHg, n=6) nor was it increased in WT mice after selective inhibition of TLR3 signaling with CU CPT 4a. Thus, the TLR3/TRIF, but not TLR4, pathway is essential for Ang II hypertension. In parallel with the pressure responses to Ang II, renal expression of Nox4 was significantly decreased in TLR3ko mice but was unchanged in TLR4-del and WT mice. In contrast to Ang II-induced hypertension, AngII-induced cardiac hypertrophy, measured as heart weight to body weight ratio, after 3 weeks of Ang II infusion was reduced in both TLR4-del (5.38 ± 0.19 mg/g) and TLR3ko (4.99 ± 0.15 mg/g) compared to WT (7.14 ± 0.49 mg/g). Thus, cardiac hypertrophy is abrogated in TLR4-del despite a robust pressor response to Ang II. The cardiac pro-inflammatory gene expression of tumor necrosis factor alpha ( Tnfa ), NADPH oxidase 4 ( Nox4 ), and matrix metalloproteinase 9 ( Mmp9 ) was increased in WT heart but attenuated in both TLR4-del and TLR3ko hearts. The results indicate a selective dependence of Ang II hypertension on TLR3/TRIF pathway whereas Ang II-induced cardiac hypertrophy depends on both TLR3 and TLR4, likely through the common TRIF adaptor protein.

The autonomic and the immune systems play major roles in the pathogenesis of cardiovascular disease and hypertension. To date, those 2 systems have been studied extensively but independently by cardiovascular biologists and by immunologists. The notion that the autonomic system can modulate the immune system and thereby influence the pathogenesis of cardiovascular disease and hypertension and their clinical outcome is novel and critical. In this brief review we focus on that interaction and an integrated understanding of the neuro-immune axis. We also highlight recent progress and future research directions. The main theme is that dysregulation of the autonomic system enhances the inflammatory response of the innate and adaptive immune systems leading to the initiation or acceleration of pathological processes and worsening of cardiovascular risks. The therapeutic potential of restoring an optimal autonomic control of the immune system is very promising. Both components of the neuro-immune axis may be involved in its disruption. One is the autonomic nervous system, which may be dysregulated or imbalanced with increased sympathetic and decreased parasympathetic activation. The other is the immune system itself, which may be abnormally sensitive to the modulatory influence of the autonomic system. These 2 components are briefly described below. #### Autonomic Dysregulation in Chronic Hypertension The complexity of cardiovascular mechanisms that contribute to hypertension has been challenging. The roles of vascular and renal abnormalities are undeniable. Changes in vasomotor tone, sodium retention, and renal renin release are all well established. For decades the contribution of the sympathetic nervous system to chronic hypertension was not fully appreciated. Its importance is now well recognized.1–5 In a 1982 review, we described the neural sites and mechanisms involved in exaggerated sympathetic nerve activity (SNA) in several animal models, as well as in human hypertension.1 Moreover, there has been a surge of data highlighting the damaging cardiovascular effects …

We have reported that angiotensin II (Ang II) induces toll-like receptors (TLRs) in the innate immune system. Here we tested which of the two major adaptor proteins of TLRs, the myeloid differentiation protein 88 (MyD88) or the TIR-domain containing adaptor inducing interferon β (TRIF) mediates the Ang II-induced responses. Infusion of Ang II (3000 ng/kg/min) subcutaneously for 3 weeks in WT mice (C57BL/6J) increased systolic blood pressure to a peak value of 147 ± 4 mm Hg and resulted in a 40% increase in heart weight to body weight ratio (HW/BW). Surprisingly, in MyD88-/- mice the pressor response was enhanced significantly to 163 ± 6 mm Hg (P

We tested the role of two distinct adaptors of toll-like receptor (TLR) signaling on Ang II-induced hypertension and cardiac hypertrophy. These TLR adaptors, myeloid differentiation protein 88 (MyD88) and TIR domain-containing adaptor inducing interferon β (TRIF) facilitate distinct inflammatory signaling pathways. In an earlier study, we reported that MyD88-/- mice are protected from cardiac hypertrophy and pro-inflammatory gene expression after myocardial infarction. Our current results with 3 weeks infusion of Ang II (3000 ng/kg/min) vs. saline indicate that in MyD88-/- mice, the pressor response to Ang II and cardiac hypertrophy were increased more than in wild type (WT) mice. In Ang II-infused WT, systolic blood pressure (SBP) peaked at 147 ± 4 mmHg whereas in Ang II-infused MyD88-/- mice SBP reached a peak value of 163 ± 6 mmHg. However, in mice with non-functional TRIF adaptor mutant (Trifmut), SBP did not increase during Ang II infusion and remained similar to the SBP in saline-infused mice (115 ± 3 mmHg). Baseline SBP was not different among WT, MyD88-/- and Trifmut mice. The increase in heart weight to body weight ratio (HW/BW) between saline and Ang II-infused mice was greater in MyD88-/- mice than WT mice (60% increase in MyD88-/- vs. 40% increase in WT), whereas it was less in Trifmut mice (22% increase). Accordingly, expression of several inflammatory genes (Tnfa, Nox4 and Agtr1a) was significantly greater (P< 0.05) in the heart and kidney of Ang II-infused MyD88-/- mice compared with Ang II-infused WT mice, whereas expression of these genes in Trifmut mice was either unchanged or reduced. We conclude that- (1) Ang II-induced hypertension, cardiac hypertrophy and inflammatory gene expression are mediated by activation of a TRIF-dependent pathway, but not by the MyD88-dependent pathways, and (2) Enhanced Ang II effects on SBP and hypertrophy in MyD88-/- mice suggest that MyD88 may serve as a negative regulator of the TRIF pathway in Ang II-induced hypertension. Selective targeting of these adaptor proteins may have significant therapeutic implications.

High fat diet inhibits the cholecystokinin (CCK) induced satiety signal in vagal afferents, which may contribute to the associated increased food intake. We hypothesized that a defect of Ca 2+ -activated chloride channel (CaCC) in high fat (HFD) fed mice leads to the reduction of CCK responses in intestinal vagal afferent nodose neurons. By using the whole cell patch-clamp in cultured nodose ganglia neurons isolated from C57BL/6 mice, we found that CCK (10nM) induced large inward chloride (Cl - ) currents (36.0±11pA/pF) that were eliminated with the fast Ca 2+ chelator BAPTA (1.0±0.5pA/pF, n=7), and reduced significantly by the CaCC channel inhibitor niflumic acid (100 μM, 13.6±2.0pA/pF, p0.05) and the mRNA of CCK receptor A was even increased from 1.06±0.37 in control to 1.54±0.41 in HFD ganglia (n=4, p2+ (20μM). We found that the maximum current was smaller in DiI labeled intestinal nodose neurons from HFD fed mice (13.7±2.9 pA/pF vs. 26.9±3.6 pA/pF in control mice, n=10, p Our results indicate that CCK-activated currents recorded from intestinal vagal afferent nodose neurons are reduced in mice fed a HFD, and are associated with reduced expression of a CCK-activated Ca 2+ -dependent Cl - channel. This mechanism may contribute significantly to HFD-induced suppression of the satiety reflex.

Our laboratory previously identified an abnormally elevated CD161a+ immune cell population in splenocytes of the pre-hypertensive Spontaneously Hypertensive Rat (SHR) that was abnormally expanded following cholinergic activation with nicotine . In the present study, we tested the hypothesis that the expanded CD161a+ cell population represents an activated monocyte/macrophage population and are present in the bone marrow (BM). We isolated cells from the spleen and BM of pre-hypertensive (4-5 week old) SHR (n=3) and age-matched normotensive Wistar Kyoto (WKY, n=3) rats. Isolated cells were stained with a fluorochrome conjugated anti-rat CD161a monoclonal antibody and analyzed by flow cytometry. CD161a+ cells were more prevalent in splenocytes and BM of the pre-hypertensive SHR, compared to WKY (8.7 ± 1.4% vs 1.2 ± 0.6% and 12.6 ± 1.8% vs 1.7 ± 0.8%, respectively, p0.05). Nicotine had no effect on the CD161a+/CD68- cell population in either splenocytes or BM cells of the SHR or WKY. Thus, an activated monocyte/macrophage population (CD161a+/CD68+) is expanded by cholinergic activation in both the BM and spleen of SHR, but only in the BM and not spleen of the WKY. Since, the pro-inflammatory nicotinic response present in the BM of the WKY is abrogated in the peripheral spleen, we conclude that the retention of this response in the SHR may contribute to the development of hypertension.

Abstract —Angiotensin II (Ang II) increases renal sympathetic nerve activity in anesthetized mice before and after ganglionic blockade, suggesting that Ang II may directly activate postganglionic sympathetic neurons. The present study directly tested this hypothesis in vitro. Neurons were dissociated from aortic-renal and celiac ganglia of C57BL/6J mice. Cytosolic Ca 2+ concentration ([Ca 2+ ] i ) was measured with ratio imaging using fura 2. Ang II increased [Ca 2+ ] i in a subpopulation of sympathetic neurons. At a concentration of 200 nmol/L, 14 (67%) of 21 neurons responded with a rise in [Ca 2+ ] i . The Ang II type 1 (AT 1 ) receptor blocker (losartan, 2 μmol/L) but not the Ang II type 2 (AT 2 ) receptor blocker (PD123,319, 4 μmol/L) blocked this effect. The Ang II–induced [Ca 2+ ] i increase was abolished by removal of extracellular Ca 2+ but not altered by depletion of intracellular Ca 2+ stores with thapsigargin. Ang II no longer elicited a [Ca 2+ ] i increase in the presence of lanthanum (25 μmol/L). The specific N-type and L-type Ca 2+ channel blockers, ω-conotoxin GVIA and nifedipine, respectively, significantly inhibited the Ang II–induced [Ca 2+ ] i increase. The protein kinase C inhibitor H7 but not the protein kinase A inhibitor H89 blocked the response to Ang II. These results demonstrate that Ang II selectively activates a subpopulation of postganglionic sympathetic neurons in aortic-renal and celiac ganglia, triggering Ca 2+ influx through voltage-gated Ca 2+ channels. This effect is mediated through AT 1 receptors and requires the activation of protein kinase C. The activation of a subgroup of sympathetic neurons by Ang II may exert unique effects on kidney function in pathological states associated with elevated Ang II.

Abstract Baroreceptor nerve endings are located in the adventitia of the carotid sinuses and aortic arch. The goal of the present study was to develop a method for gene transfer to the carotid sinus adventitia. Replication-deficient adenovirus containing the gene for Escherichia coli β-galactosidase (β-Gal) was applied topically to the carotid sinuses of anesthetized rabbits. Transgene expression was localized by histochemical staining and quantified by chemiluminescence assay (Galacto-Light). Possible effects of adenovirus on baroreceptor sensitivity were investigated by recording baroreceptor activity from the vascularly isolated carotid sinus over a pressure range of 0 to 160 mm Hg. β-Gal expression in carotid sinus was evident 1 day after virus application, was dose dependent, and was markedly enhanced after 4 days. Expression was restricted to the adventitia of the vessel wall and was not present in vehicle-treated carotid sinuses. Baroreceptor sensitivity measured from carotid sinuses exposed to adenovirus 4 to 5 days beforehand was not altered compared with that measured from control carotid sinuses. In summary, topical application of adenoviral vectors to the carotid sinus provides transgene expression restricted to the region of baroreceptor innervation. The technique provides a novel approach to delineate mechanisms involved in baroreceptor activation and to deliver neuroactive gene products to the baroreceptors.

Toll-like receptors (TLR) are a class of pattern-recognizing receptors (PRR) that play a central role in the innate immune response during infection and sterile injury. MyD88 is an adapter protein that mediates the majority of TLR responses. Since inflammation is a coexisting condition in several cardiovascular diseases, TLRs are thought to play a major role in these conditions. We have previously shown that indeed post-MI survival was significantly improved and cardiac fibrosis and hypertrophy were reduced in MyD88-/- mice (Singh et al. 2012. JMCC). In this study we tested whether angiotensin II (AngII) hypertension and cardiac hypertrophy depended on TLR signaling pathways mediated by MyD88. Male MyD88-/- mice and C57BL/6 mice of 10 to 12 weeks of age were subcutaneously implanted with osmotic minipumps (Alzet) eluting saline or AngII (Sigma, 733 ng/kg/min). Tailcuff pressures were measured with BP2000 (Visitech Systems). Mice were sacrificed after 3 weeks of AngII infusion and hearts were collected for weighing and gene expression analyses. In control WT mice, mean arterial pressure (MAP) remained near baseline levels during the first week of infusion averaging 71± 2 mm Hg and then increased to an average of 100 ± 3 mm Hg and 102 ± 2 mm Hg during the 2nd and 3rd weeks of infusions, respectively. In MyD88-/- mice, MAP increased from a baseline of 84 ± 3 to a high of 120 ± 7 mm Hg during the first week and then declined to 109 ± 6 and 91 ± 9 mm Hg during the 2nd and 3rd weeks. The heart weight to body weight ratios (HW/BW x 1000) after 3 weeks of AngII infusion were not significantly different between WT (5.23 ± 0.15) and MyD88-/- (4.76 ± 0.16). Increases in cardiac hypertrophic marker gene Acta1 were up 4-fold in both WT and MyD88-/- mice, and increases in proinflammatory TNF-alpha, IL-1β, and Nox4 were seen in both genotypes, but the increase in TNF-alpha was significantly greater in MyD88-/- mice. We conclude that unlike post- MI cardiac hypertrophy, the AngII cardiac hypertrophy is not MyD88 -dependent, yet the delayed Ang II pressor response is abrogated in the absence of MyD88. The delayed pressor response to AngII infusion is likely dependent on the immune system.

Sarcoglycan mutations cause muscular dystrophy in humans. We recently demonstrated that sarcoglycan delta deficient (Sgcd-/-) mice with muscular dystrophy exhibit autonomic dysregulation [Hypertension 2010]. We hypothesized that excessive sympathetic activity contributes to skeletal muscle pathology, decreased locomotor activity and autonomic dysregulation in young (10-12 wks) Sgcd-/- mice. The centrally-acting sympathoinhibitory drug rilmenidine (RIL) was infused into the brain of control C57BL6 and Sgcd-/- mice by osmotic pump for 7-9 wks beginning at 3 wks of age (42 ng/g/hr, ICV). Separate groups of mice were infused with saline vehicle (VEH). Blood pressure (BP), heart rate (HR) and locomotor activity were measured by telemetry. Cardiac (HR responses to propranolol) and vasomotor (BP response to ganglionic blockade) sympathetic tone were increased in VEH-treated Sgcd-/- mice, and normalized by RIL (Table). The RIL-induced sympathoinhibition in Sgcd-/- mice was accompanied by increases in baroreflex sensitivity (BRS, sequence technique), cardiovagal tone (HR response to atropine) and activity, with no change in BP (Table). RIL also decreased oxidative stress (superoxide) by 56% and fibrosis in Sgcd-/- skeletal muscle. RIL did not affect measured variables in control mice (Table). In summary, RIL-induced sympathoinhibition decreased skeletal muscle pathology, increased locomotor activity and improved autonomic regulation in young Sgcd-/- mice. The results implicate increased sympathetic activity in the pathogenesis of muscular dystrophy, and suggest that targeting the brain to inhibit sympathetic activity may provide a novel therapeutic approach.

Abstract Structural changes in large arteries are often considered the predominant mechanism responsible for decreased baroreflex sensitivity and baroreceptor resetting in hypertension, atherosclerosis, and aging. Recent work has demonstrated that “functional” mechanisms, both at the level of the peripheral sensory endings and within the central nervous system, contribute significantly to altered baroreflex responses. We have conducted both reductive studies of mechanoelectrical transduction in cultured baroreceptor neurons and integrative studies with in vivo recordings of the activity of baroreceptor afferent fibers and efferent sympathetic nerves. Results suggest that the primary mechanism of mechanical activation of baroreceptor neurons involves opening of stretch-activated ion channels susceptible to blockade by gadolinium. Baroreceptor nerve activity is modulated by the activity of potassium channels and the sodium-potassium pump and by paracrine factors, including prostacyclin, oxygen free radicals, and factors released from aggregating platelets. Endothelial dysfunction and altered release of these paracrine factors contribute significantly to the decreased baroreceptor sensitivity in hypertension and atherosclerosis. The central mediation of the baroreflex depends on the pulse phasic pattern of afferent baroreceptor discharge. Baroreflex-mediated inhibition of sympathetic nerve activity is well maintained during pulse phasic afferent activity. Continuous, nonphasic baroreceptor discharge or a rapid (>1.5 Hz) pulse phasic discharge results in disinhibition of sympathetic activity. This disinhibition during continuous baroreceptor input is exaggerated with aging. Thus, a defect in central mediation of the baroreflex may be a major cause of the impaired baroreflex and sympathoexcitation in the elderly. In summary, functional neural mechanisms, in addition to structural vascular changes, contribute importantly to altered baroreflex responses in normal and pathophysiological states. Therefore, it may be possible to implement therapies that reverse functional changes and restore baroreflex sensitivity long before the reversal of structural vascular changes.

Abstract The goal of this study was to determine whether nitric oxide (NO) and the NO donor, S -nitrosocysteine (cysNO), modulate the activity of carotid sinus baroreceptors. Baroreceptor activity was recorded from the vascularly isolated carotid sinus in anesthetized rabbits. Baroreceptor activity decreased in a dose-dependent manner after injection of either NO or cysNO as constant pressure was maintained, and activity recovered spontaneously over time, within seconds to minutes. The baroreceptor pressure-activity relation was shifted significantly to the right by cysNO, with a profound suppression of activity at high pressure. Baroreceptor activity at 160 mm Hg averaged 76±8%, 60±6%, and 36±5% of the control maximum during exposure to 10 −4 , 2 to 3×10 −4 , and 10 −3 mol/L cysNO, respectively. The inhibition of activity by the l and d isomers of cysNO was equivalent and was blocked by reduced hemoglobin, suggesting that the effect was mediated by NO. The suppression of baroreceptor activity by cysNO was not related to vascular relaxation as measured by videomicrometer. Inhibition of soluble guanylate cyclase with methylene blue or 6-anilinoquinoline-5,8-quinone (LY83583, 10 −5 mol/L) did not attenuate and dibutyryl cGMP (10 −3 mol/L) did not mimic the suppression of baroreceptor activity by cysNO, suggesting a cGMP-independent mechanism. Activation of endogenous NO formation with thimerosal (10 −5 to 10 −4 mol/L) reduced maximum baroreceptor activity in five of eight experiments to 59±7% of the control maximum. The NO synthase inhibitor nitro- l -arginine methyl ester (L-NAME, 10 −4 mol/L) by itself failed to influence baroreceptor activity but prevented thimerosal-induced suppression of activity. Addition of l -arginine (10 −3 mol/L) after L-NAME restored the inhibitory influence of thimerosal. The results indicate that NO and cysNO suppress baroreceptor activity through a mechanism independent of guanylate cyclase activation and vascular relaxation and that endogenous NO released by chemical activation suppresses baroreceptor activity.

Muscular dystrophy is a catastrophic, fatal neuromuscular disease in need of new therapies. Angiotensin II binding to AT 1 receptors (AT 1 R) contributes to fibrosis in dystrophic skeletal muscle and AT 1 R blockers are currently being tested in clinical trials. Recently, we reported that autonomic dysregulation precedes and predicts development of cardiomyopathy in sarcoglycan delta deficient (Sgcd-/-) mice with muscular dystrophy (Clin Auton Res, 2010 ). We hypothesized that infusion of the angiotensin peptide Ang-[1-7] will rescue skeletal muscle, locomotor, and autonomic nervous system phenotypes in young Sgcd-/- mice. Control and Sgcd-/- mice were infused with Ang-[1-7] (300 ng/kg/min) for 8 wks beginning at 3 wks of age. Blood pressure (BP), heart rate (HR) and activity were recorded by telemetry in treated and untreated mice. Baroreflex sensitivity (BRS, sequence technique) and resting cardiac vagal and sympathetic tone (HR responses to atropine and propranolol) were measured. BP, activity, BRS and vagal tone were lower in Sgcd-/- vs. control mice, whereas sympathetic tone was higher (Table). Ang-[1-7] normalized activity, BRS, and sympathovagal balance in Sgcd-/- mice without affecting BP, and did not influence any variable in control mice. Skeletal muscle fibrosis present in Sgcd-/- mice (18±1%, n=9) was markedly reduced by Ang-[1-7] (3±1%, n=5). We conclude that Ang-[1-7] reduces skeletal muscle fibrosis and restores locomotor activity and sympathovagal balance in Sgcd-/- mice, without lowering BP. Ang-[1-7] and/or enhancement of its endogenous production may provide a novel therapeutic approach to muscular dystrophy.

pH sensitivity has been rarely studied in vagal afferents of nodose ganglia (NG) neurons. Using whole-cell patch-clamp technique in isolated NG neurons, we recently identified a pH-conditioned Cl - current (pH-I) that was evoked following 2 or 3 brief (10s) exposures to low extracellular pH (7.0-6.0) in 16 of 22 (70%) cells (FASEB J, 2012). The current is large (904.3±159.9pA) and prolonged, lasting 10∼15 minutes, and causes significant depolarization (Δ35.2±4.4mV). In the present study, we tested the hypothesis that reactive oxygen species (ROS) mediates this pH-conditioned Cl - conductance. We found that the rate of increase in fluorescence [(F-F 0 )/F 0 ] of NG neurons loaded with dihydroethidine (ROS dye) rose dramatically following the brief exposures to pH 6.0 from a control of 0.04±0.01 to 0.12±0.02 units/min over 10∼15 minutes (n=31 neurons, p2 O 2 induced currents that mimicked the pH conditioned currents. Because of similarities between the pH-conditioned Cl - conductance and the previously described “swell-activated” Cl - current induced with hypoosmotic solutions, we superfused the NG neurons with the H 2 O 2 scavenger PEG-catalase (1000 units/ml). PEG-catalase blocked significantly (p2 O 2 . Opening of these outward Cl - conductances that cause sustained depolarization of vagal afferents may induce a beneficial reflex sympathoinhibition during myocardial ischemia/acidosis or initiate a gastro-intestinal post-prandial satiety reflex.

Immune cells from patients with essential hypertension are hyper-responsive to activation of toll-like receptor (TLR) 4 with lipopolysaccharide. Activation of the alpha7 nicotinic cholinergic receptor exerts an anti-inflammatory effect on innate immune cells. We hypothesized that this anti-inflammatory cholinergic response is lacking in the Spontaneously Hypertensive Rat (SHR) and recently reported that nicotine suppresses TLR7 and TLR9 mediated IL-6 secretion by isolated splenocytes of WKY rats; whereas, it dramatically enhances IL-6 secretion in SHR. Here we aimed to confirm these effects in vivo and identify potential cellular mediators of the observed immune responses. Flow cytometry on splenocytes revealed a greater number of CD3-/CD8dim innate immune cells in SHR (4.0±0.56%), compared to WKY (2.97±0.6%)(n=3, pIn vivo, we infused saline or nicotine (15mg/kg/day) over 24 hours using subcutaneous pumps. At 20 hrs, animals received an intraperitoneal injection of a TLR7 ligand (Clo97). Serum was collected at the end of the 24 hr infusion. Similar to previous in vitro results, nicotine suppressed the serum IL-6 response to i.p. Clo97 from 108±28 pg/ml in WKY (n=4) to 12±7 pg/ml (p

Aggregating platelets release factors that act in a local paracrine manner to alter vascular tone. The purpose of the present study was to explore the possibility that factors released from aggregating platelets may alter the sensitivity of arterial baroreceptors. Baroreceptor activity was recorded from the vascularly isolated carotid sinus of rabbits anesthetized with sodium pentobarbital. The carotid sinus was filled with oxygenated Krebs-Henseleit buffer and distended with slow ramp increases in nonpulsatile pressure. Sensitivity of baroreceptors to increased pressure was determined before and during intraluminal exposure of the sinus to washed human platelets suspended in Krebs' buffer. Platelets activated with thrombin (0.4 units/ml) decreased baroreceptor activity and the slope of the pressure-activity curve significantly (n = 6). The platelet-induced decrease in baroreceptor sensitivity was related to the duration of exposure to platelets with no change in baroreceptor activity after 4 minutes and a progressive decrease in activity over the next 12 minutes. The slope of the pressure-nerve activity relation averaged 1.26 +/- 0.08 %/mm Hg during control and decreased to 0.97 +/- 0.22, 0.80 +/- 0.19, and 0.53 +/- 0.15 %/mm Hg after 12-16 minutes of exposure to 10(7), 10(8), and 3-6 x 10(8) activated platelets/ml, respectively (p less than 0.05). Baroreceptor sensitivity was restored after removal of platelets from the carotid sinus. Thrombin alone had no effect on baroreceptor sensitivity. Activated platelets did not alter the carotid pressure-diameter relation, suggesting a direct inhibitory effect on baroreceptors. The slope of the pressure-activity curve and maximum baroreceptor activity were not decreased by the stable thromboxane analogue U46619, serotonin, or ADP.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to test the hypothesis that prostaglandins released from vascular endothelial cells contribute to activation of baroreceptors during increases in arterial pressure. Baroreceptor activity was recorded from the vascularly isolated carotid sinus in rabbits anesthetized with chloralose. Baroreceptor activity was measured during ramp or step increases in nonpulsatile carotid sinus pressure over a range of 0-175 mm Hg. Exposure of the isolated carotid sinus to inhibitors of prostaglandin formation (indomethacin [n = 10] or aspirin [n = 6]) decreased baroreceptor activity significantly (p less than 0.05). The slope of the pressure-activity relation averaged 0.80 +/- 0.07 %/mm Hg (mean +/- SEM) during control measurements and 0.72 +/- 0.06 and 0.63 +/- 0.05 %/mm Hg during exposure to 10 and 20 microM indomethacin, respectively. Exposure of the carotid sinus to exogenous prostacyclin (PGI2 [n = 11]) increased baroreceptor activity significantly. The slope of the pressure-activity relation averaged 0.89 +/- 0.10, 1.09 +/- 0.09, and 1.26 +/- 0.16 %/mm Hg during control and during exposure to 10 and 20 microM PGI2, respectively. Activity returned to control after removal of PGI2 (0.89 +/- 0.12 %/mm Hg). Removal of endothelium with either a balloon catheter (n = 4) or a jet of a 95% O2-5% CO2 gas mixture (n = 6) decreased the slope of the pressure-activity relation from 0.92 +/- 0.09 to 0.56 +/- 0.08 %/mm Hg (p less than 0.05). Exposure of the denuded sinus to exogenous PGI2 (20 microM [n = 4]) restored activity (slope = 1.09 +/- 0.24 %/mm Hg). Neither indomethacin (n = 5) nor PGI2 (n = 5) nor denudation (n = 5) significantly altered the pressure-diameter relation of the carotid sinus (sonomicrometers), suggesting that the effects on baroreceptor discharge are not caused by altered stretch of the carotid sinus at a given pressure. The results suggest that prostaglandins (e.g., PGI2) released from endothelium contribute in a paracrine manner to activation of baroreceptors during increases in arterial pressure.

The molecular mechanism of pH sensing by chemoreceptors is not clear, although it had been proposed to be mediated by a drop in intracellular pH of carotid body glomus cells, which inhibits a K + current. Recently, pH-sensitive ion channels have been described in glomus cells that respond directly to extracellular acidosis. In this study, we investigated the possible molecular mechanisms of carotid body pH-sensing by recording the responses of glomus cells isolated from rat carotid body to rapid changes in extracellular pH using whole-cell patch-clamping technique. Extracellular acidosis evoked transient inward currents in glomus cells that were evident at pH 7.0 and half-activated (pH 50) at 6.3. The current had the characteristics of ASICs. It averaged 40.7±15.7 pA (n=5) at pH 5.0 and was blocked by the ASIC channel blocker amiloride (200 μm) to 2.5±1.6 pA. Na + free bathing solution eliminated the current and a Ca 2+ free buffer enhanced (P+ from the pipette solution which reduced significantly intracellular K + . This sustained depolarization was partially blocked by the TASK channels blockers anandamide (from 14.9±1.6 mV to 9.3±2.2 mV at pH 6.0, n=5) and quinidine (from 27.5±2.2 mV to 11.3±2.3 mV at pH 6.0, n=3). The results provide the first evidence that ASICs may contribute to chemotransduction of low pH by carotid body chemoreceptors, and that extracellular acidosis directly activates carotid body chemoreceptors through both ASIC and TASK channels.

The purpose of this study was to contrast the effects of static and pulsatile pressure on carotid baroreceptor activity over a wide range of mean arterial pressure. Static and pulsatile pressure were applied to the isolated carotid sinus of dogs anesthetized with chloralose. Recordings were obtained from single baroreceptor units as well as from the whole sinus nerve or a large strand of the nerve. Three observations are reported. First, in single units the pulsatile pressure threshold, which averaged 48 +/- 8 (SEM) mm Hg, was far below the static pressure threshold, which averaged 79 +/- 8 mm Hg (p less than 0.05, n = 15). Thus, pulsatility decreased the threshold by an average of 31 mm Hg in contrast to the minimal or lack of decrease in threshold reported by others in aortic baroreceptors. Second, at moderate arterial pressures a shift from static to pulsatile pressure caused a decrease in single and multiple unit activities. In single units, the decrease approximated 15% (from 42.0 +/- 2.1 to 35.5 +/- 1.9 spikes/sec, p less than 0.05, n = 25). In all units, there was no diastolic nerve activity ("silence") when diastolic pressure was 1 to 10 mm Hg above static pressure threshold; 80% of the units exhibited "diastolic silence" when diastolic pressure was 20-30 mm Hg above threshold and 40% of the units showed silence at diastolic pressures 40-50 mm Hg above threshold. In whole nerve recordings, pulsatility increased activity from 57 +/- 15 to 142 +/- 29 spikes/sec (p less than 0.05) at low mean arterial pressures (50 and 75 mm Hg), as expected from the reduction in pressure threshold noted in single units, and decreased activity by approximately 15% (from 373 +/- 69 to 320 +/- 55 spikes/sec, p less than 0.05, n = 9) at mean arterial pressures of 125 and 150 mm Hg. This decrease in activity with a shift from static to pulsatile pressure at moderate arterial pressures has not been reported previously. Third, the static pressure-activity curve was sigmoid, and its gain peaked sharply at 75-100 mm Hg; in contrast, the pulsatile pressure-activity curve was linear between 25 and 150 mm Hg, and its maximum gain was half the maximum gain during static pressure. These differences between the static pressure-activity curve and the pulsatile pressure-activity curve were noted during both increases and decreases in carotid sinus pressure; both curves exhibited some hysteresis during the decreases in pressure.

The threshold pressure of single baroreceptor units is decreased after compared with before exposure to pulsatile pressure according to previous studies in our laboratory. The purpose of the present study is to characterize the determinants of sensitization of arterial baroreceptors by pulsatile pressure. Carotid sinus nerve activity was recorded in dogs anesthetized with chloralose. Two indexes of baroreceptor "sensitivity" were obtained by comparing nerve activity before and immediately after exposure of the isolated carotid sinus to pulsatile pressure for periods up to 10 minutes. Sensitization occurred 1) when the threshold pressure of single baroreceptor units determined with a slow nonpulsatile ramp decreased after as compared with before pulsing and 2) when multiple unit activity increased after as compared with before pulsing at various mean levels of static pressure. Sensitization was evident after pulsing at mean pressure of 50 and 100 mm Hg, but not at 150 and 200 mm Hg, and was caused by the pulsatile change in diameter or deformation and not by the pulsatile change in wall tension. The magnitude of the effect was directly related to the duration of the pulsing period and to the frequency and amplitude of the pressure pulses. The sensitization could not be explained by increased diameter (sonomicrometers) or strain of the carotid sinus at the same pressure after pulsing; thus, there was an increase in "strain sensitivity" that outlasted the period of pulsing by up to several minutes. In most experiments the shift from static to pulsatile pressure at 50 and 100 mm Hg caused an increase in nerve activity, yet sensitization occurred after pulsing when one would have expected postexcitatory hyperpolarization or depression of activity upon return to static pressure. The sensitization was not caused by the release of prostacyclin from the endothelium since it was not reduced after endothelial denudation or inhibition of cyclooxygenase with indomethacin (30-80 microM) or ibuprofen (250 microM). We speculate that sensitization of baroreceptors by pulsatile pressure may contribute to the decreased sympathetic activity after periods of elevated pulse pressure (e.g., after exercise). We also propose that the decreased sensitivity of baroreceptors after acute elevation of arterial pressure (acute resetting) may be offset in part by the sensitizing effect of increased pulsatile stretch.

High-frequency positive-pressure ventilation (HFPPV) was compared to intermittent positive-pressure ventilation (IPPV) during unilateral atelectasis with and without halothane anesthesia. Dogs with electromagnetic flow probes chronically implanted on their main (Qt) and left (Ql) pulmonary arteries were ventilated via Carlen's dual-lumen endotracheal tubes. In eight closed-chest dogs, about 43% of the cardiac output perfused the left lung during bilateral ventilation by either a Harvard animal respirator (IPPV) or a Health-dyne model 300 high-frequency ventilator (HFPPV). Unilateral atelectasis decreased blood flow (Ql/Qt) to that lung. Ql/Qt was 19 +/- 1% with HFPPV during left-lung atelectasis and right-lung ventilation, compared to 32 +/- 1% with unilateral IPPV. This suggests that HFPPV permits stronger hypoxic pulmonary vasoconstriction. Addition of 1% halothane increased blood flow to the atelectatic left lung during unilateral ventilation with IPPV but not with HFPPV. This suggests that halothane decreases the effects of hypoxic pulmonary vasoconstriction during conventional ventilation but not during HFPPV.