Vasodilators activate the anion channel TMEM16A in endothelial cells to reduce blood pressure.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl-) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca2+)-activated Cl- currents in endothelial cells from control (TMEM16Afl/fl) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell-specific knockout of TMEM16A (TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca2+ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca2+ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca2+-dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Vasodilators activate TMEM16A channels in endothelial cells to reduce blood pressure.

Endothelial cells (ECs) regulate vascular contractility to control regional organ blood flow and systemic blood pressure. Several cation channels are expressed in ECs which regulate arterial contractility. In contrast, the molecular identity and physiological functions of anion channels in ECs is unclear. Here, we generated tamoxifen-inducible, EC-specific TMEM16A knockout ( TMEM16A ecKO) mice to investigate the functional significance of this chloride (Cl - ) channel in the resistance vasculature. Our data demonstrate that TMEM16A channels generate calcium-activated Cl - currents in ECs of control ( TMEM16A fl/fl ) mice that are absent in ECs of TMEM16A ecKO mice. Acetylcholine (ACh), a muscarinic receptor agonist, and GSK101, a TRPV4 agonist, activate TMEM16A currents in ECs. Single molecule localization microscopy data indicate that surface TMEM16A and TRPV4 clusters locate in very close nanoscale proximity, with ∼18% exhibiting overlap in ECs. ACh stimulates TMEM16A currents by activating Ca 2+ influx through surface TRPV4 channels without altering the size or density of TMEM16A or TRPV4 surface clusters, their spatial proximity or colocalization. ACh-induced activation of TMEM16A channels in ECs produces hyperpolarization in pressurized arteries. ACh, GSK101 and intraluminal ATP, another vasodilator, all dilate pressurized arteries through TMEM16A channel activation in ECs. Furthermore, EC-specific knockout of TMEM16A channels elevates systemic blood pressure in conscious mice. In summary, these data indicate that vasodilators stimulate TRPV4 channels, leading to Ca 2+ -dependent activation of nearby TMEM16A channels in ECs to produce arterial hyperpolarization, vasodilation and a reduction in blood pressure. We identify TMEM16A as an anion channel present in ECs that regulates arterial contractility and blood pressure. One sentence summary: Vasodilators stimulate TRPV4 channels, leading to calcium-dependent activation of nearby TMEM16A channels in ECs to produce arterial hyperpolarization, vasodilation and a reduction in blood pressure.

Ovariectomy Via 12/15-lipoxygenase Augments Angiotensin II-Induced Hypertension and Its Pathogenesis in Female Mice.

BACKGROUND: Ang II (angiotensin II) releases arachidonic acid from tissue phospholipids that is metabolized by 12/15-lipoxygenase (ALOX15), generating 12(S)- and 15(S)-hydroxyeicosatetraenoic acid (HETE), which have been implicated in cardiovascular and renal diseases. In this study, we tested the hypothesis that ovariectomy augments Ang II-induced hypertension and renal pathophysiological changes via ALOX15 activation in female mice. METHODS: Ang II (700 ng/kg/min) was infused subcutaneously by osmotic pumps for 2 weeks in intact and ovariectomized wild-type and Alox15 knockout (ALOX15KO) female mice for evaluation of hypertension and associated pathogenesis. RESULTS: Ang II increased blood pressure, impaired autonomic function, and increased renal reactive oxygen species production and plasma 12(S)-HETE level without altering renal function in intact wild-type mice. However, in OVX-wild-type mice with depleted plasma 17ß-estradiol, the effects of Ang II on blood pressure, autonomic impairment, renal reactive oxygen species production, and plasma 12(S)- but not 15(S)-HETE was markedly enhanced. In OVX-wild-type mice, Ang II also increased renal alox15 mRNA, urine 12(S)-HETE, water intake, urine output, decreased osmolality, increased urinary excretion of vasopressin prosegment copeptin, protein/creatinine ratio, and caused renal hypertrophy, fibrosis, and inflammation. These effects of Ang II were attenuated in ALOX15KO mice. CONCLUSIONS: These data suggest that 17ß-estradiol protects against Ang II-induced hypertension and associated pathogenesis in female mice, most likely via inhibition of ALOX15-arachidonic acid derived production of 12(S)-HETE. Therefore, the selective inhibitors of ALOX15 or 12(S)-HETE receptor antagonists could be useful for treating hypertension and its pathogenesis in postmenopausal, hypoestrogenic women, or females with ovarian failure.

White matter damage as a consequence of vascular dysfunction in a spontaneous mouse model of chronic mild chronic hypoperfusion with eNOS deficiency.

Vascular cognitive impairment and dementia (VCID) is the second most common form of dementia after Alzheimer's disease (AD). Currently, the mechanistic insights into the evolution and progression of VCID remain elusive. White matter change represents an invariant feature. Compelling clinical neuroimaging and pathological evidence suggest a link between white matter changes and neurodegeneration. Our prior study detected hypoperfused lesions in mice with partial deficiency of endothelial nitric oxide (eNOS) at very young age, precisely matching to those hypoperfused areas identified in preclinical AD patients. White matter tracts are particularly susceptible to the vascular damage induced by chronic hypoperfusion. Using immunohistochemistry, we detected severe demyelination in the middle-aged eNOS-deficient mice. The demyelinated areas were confined to cortical and subcortical areas including the corpus callosum and hippocampus. The intensity of demyelination correlated with behavioral deficits of gait and associative recognition memory performances. By Evans blue angiography, we detected blood-brain barrier (BBB) leakage as another early pathological change affecting frontal and parietal cortex in eNOS-deficient mice. Sodium nitrate fortified drinking water provided to young and middle-aged eNOS-deficient mice completely prevented non-perfusion, BBB leakage, and white matter pathology, indicating that impaired endothelium-derived NO signaling may have caused these pathological events. Furthermore, genome-wide transcriptomic analysis revealed altered gene clusters most related to mitochondrial respiratory pathways selectively in the white matter of young eNOS-deficient mice. Using eNOS-deficient mice, we identified BBB breakdown and hypoperfusion as the two earliest pathological events, resulting from insufficient vascular NO signaling. We speculate that the compromised BBB and mild chronic hypoperfusion trigger vascular damage, along with oxidative stress and astrogliosis, accounting for the white matter pathological changes in the eNOS-deficient mouse model. We conclude that eNOS-deficient mice represent an ideal spontaneous evolving model for studying the earliest events leading to white matter changes, which will be instrumental to future therapeutic testing of drug candidates and for targeting novel/specific vascular mechanisms contributing to VCID and AD.

Activation of GPR75 Signaling Pathway Contributes to the Effect of a 20-HETE Mimetic, 5,14-HEDGE, to Prevent Hypotensive and Tachycardic Responses to Lipopolysaccharide in a Rat Model of Septic Shock.

ABSTRACT: The orphan receptor, G protein-coupled receptor (GPR) 75, which has been shown to mediate various effects of 20-hydroxyeicosatetraenoic acid (20-HETE), is considered as a therapeutic target in the treatment of cardiovascular diseases in which changes in the production of 20-HETE play a key role in their pathogenesis. Our previous studies showed that 20-HETE mimetic, N -(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), protects against vascular hyporeactivity, hypotension, tachycardia, and arterial inflammation induced by lipopolysaccharide (LPS) in rats. This study tested the hypothesis that the GPR75 signaling pathway mediates these effects of 5,14-HEDGE in response to systemic exposure to LPS. Mean arterial pressure reduced by 33 mm Hg, and heart rate increased by 102 beats/min at 4 hours following LPS injection. Coimmunoprecipitation studies demonstrated that (1) the dissociation of GPR75/Gα q/11 and GPR kinase interactor 1 (GIT1)/protein kinase C (PKC) α, the association of GPR75/GIT1, large conductance voltage and calcium-activated potassium subunit ß (MaxiKß)/PKCα, MaxiKß/proto-oncogene tyrosine-protein kinase (c-Src), and epidermal growth factor receptor (EGFR)/c-Src, MaxiKß, and EGFR tyrosine phosphorylation were decreased, and (2) the association of GIT1/c-Src was increased in the arterial tissues of rats treated with LPS. The LPS-induced changes were prevented by 5,14-HEDGE. N -[20-Hydroxyeicosa-6( Z ),15( Z )-dienoyl]glycine, a 20-HETE antagonist, reversed the effects of 5,14-HEDGE in the arterial tissues of LPS-treated rats. Thus, similar to 20-HETE, by binding to GPR75 and activating the Gα q/11 /PKCα/MaxiKß, GIT1/PKCα/MaxiKß, GIT1/c-Src/MaxiKß, and GIT1/c-Src/EGFR signaling pathways, 5,14-HEDGE may exert its protective effects against LPS-induced hypotension and tachycardia associated with vascular hyporeactivity and arterial inflammation.

A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation.

Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms, and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca2+-dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and via the activation of endothelial nitric oxide synthase (eNOS) and intermediate (IK)- and small (SK)-conductance Ca2+-activated K+ channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 (Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates nonselective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility through the formation of an interdependent signaling complex with PC-2 in ECs. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to eNOS, IK channel, and SK channel activation, vasodilation, and a reduction in blood pressure.

Pharmacological Inhibition of Mammalian Target of Rapamycin Attenuates Deoxycorticosterone Acetate Salt-Induced Hypertension and Related Pathophysiology: Regulation of Oxidative Stress, Inflammation, and Cardiovascular Hypertrophy in Male Rats.

ABSTRACT: The present study aimed to explore the contribution of mammalian target of rapamycin (mTOR) in deoxycorticosterone acetate (DOCA) salt-induced hypertension and related pathophysiological changes in cardiovascular and renal tissues. DOCA salt loading resulted in an increase in systolic blood pressure, diastolic blood pressure, and mean blood pressure along with the activity of ribosomal protein S6, the effector protein of mTOR. Treatment with rapamycin, the selective inhibitor of mTOR, initiated at the fourth week of DOCA- salt administration normalized the systolic blood pressure and attenuated ribosomal protein S6 activity in the heart, aorta, and kidney. Cardiac and vascular hypertrophy, oxidative stress, and infiltration of macrophages (CD68+), the marker of inflammation, were also reduced in rapamycin-treated, DOCA-salt, hypertensive rats. In addition, renal hypertrophy and dysfunction were also reduced with rapamycin-treated hypertensive rats. Moreover, these pathophysiological changes in DOCA-salt hypertensive rats were associated with increased NADPH oxidase (NOX) activity, gp91phox (formerly NOX2) expression, ERK1/2, and p38 MAPK activities in the heart, aorta, and kidney were minimized by rapamycin. These data indicate that mTOR plays an important role in regulating blood pressure and the development of cardiovascular and renal pathophysiological changes, most likely due to increased NOX expression/activity, ERK1/2, and p38 MAPK activity with macrophages infiltration in the heart, kidney, and aorta. Pharmacological inhibition of mTOR and related signaling pathways could serve as a novel target for the treatment of hypertension.

Renin-Angiotensin System Alterations in the Human Alzheimer's Disease Brain.

BACKGROUND: Understanding Alzheimer's disease (AD) in terms of its various pathophysiological pathways is essential to unravel the complex nature of the disease process and identify potential therapeutic targets. The renin-angiotensin system (RAS) has been implicated in several brain diseases, including traumatic brain injury, ischemic stroke, and AD. OBJECTIVE: This study was designed to evaluate the protein expression levels of RAS components in postmortem cortical and hippocampal brain samples obtained from AD versus non-AD individuals. METHODS: We analyzed RAS components in the cortex and hippocampus of postmortem human brain samples by western blotting and immunohistochemical techniques in comparison with age-matched non-demented controls. RESULTS: The expression of AT1R increased in the hippocampus, whereas AT2R expression remained almost unchanged in the cortical and hippocampal regions of AD compared to non-AD brains. The Mas receptor was downregulated in the hippocampus. We also detected slight reductions in ACE-1 protein levels in both the cortex and hippocampus of AD brains, with minor elevations in ACE-2 in the cortex. We did not find remarkable differences in the protein levels of angiotensinogen and Ang II in either the cortex or hippocampus of AD brains, whereas we observed a considerable increase in the expression of brain-derived neurotrophic factor in the hippocampus. CONCLUSION: The current findings support the significant contribution of RAS components in AD pathogenesis, further suggesting that strategies focusing on the AT1R and AT2R pathways may lead to novel therapies for the management of AD.

2-Methoxyestradiol Ameliorates Angiotensin II-Induced Hypertension by Inhibiting Cytosolic Phospholipase A2α Activity in Female Mice.

[Figure: see text].

6ß-Hydroxytestosterone Promotes Angiotensin II-Induced Hypertension via Enhanced Cytosolic Phospholipase A2α Activity.

[Figure: see text].

Testosterone Metabolite 6ß-Hydroxytestosterone Contributes to Angiotensin II-Induced Abdominal Aortic Aneurysms in Apoe-/- Male Mice.

Background Sex is a prominent risk factor for abdominal aortic aneurysms (AAAs), and angiotensin II (Ang II) induces AAA formation to a greater degree in male than in female mice. We previously reported that cytochrome P450 1B1 contributes to the development of hypertension, as well as AAAs, in male mice. We also found that a cytochrome P450 1B1-generated metabolite of testosterone, 6ß-hydroxytestosterone (6ß-OHT), contributes to Ang II-induced hypertension and associated cardiovascular and renal pathogenesis in male mice. The current study was conducted to determine the contribution of 6ß-OHT to Ang II-induced AAA development in Apoe-/- male mice. Methods and Results Intact or castrated Apoe-/-/Cyp1b1+/+ and Apoe-/-/Cyp1b1-/- male mice were infused with Ang II or its vehicle for 28 days, and administered 6ß-OHT every third day for the duration of the experiment. Abdominal aortas were then evaluated for development of AAAs. We observed a significant increase in the incidence and severity of AAAs in intact Ang II-infused Apoe-/-/Cyp1b1+/+ mice, compared with vehicle-treated mice, which were minimized in castrated Apoe-/-/Cyp1b1+/+ and intact Apoe-/-/Cyp1b1-/- mice infused with Ang II. Treatment with 6ß-OHT significantly restored the incidence and severity of AAAs in Ang II-infused castrated Apoe-/-/Cyp1b1+/+ and intact Apoe-/-/Cyp1b1-/- mice. However, administration of testosterone failed to increase AAA incidence and severity in Ang II-infused intact Apoe-/-/Cyp1b1-/- mice. Conclusions Our results indicate that the testosterone-cytochrome P450 1B1-generated metabolite 6ß-OHT contributes to Ang II-induced AAA development in Apoe-/- male mice.

mTOR inhibition as a possible pharmacological target in the management of systemic inflammatory response and associated neuroinflammation by lipopolysaccharide challenge in rats.

Neuroinflammation plays a critical role during sepsis triggered by microglial activation. Mammalian target of rapamycin (mTOR) has gained attraction in neuroinflammation, however, the mechanism remains unclear. Our goal was to assess the effects of mTOR inhibition by rapamycin on inflammation, microglial activation, oxidative stress, and apoptosis associated with the changes in the inhibitor-κB (IκB)-α/nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α) pathway activity following a systemic challenge with lipopolysaccharide (LPS). Rats received saline (10 mL/kg), LPS (10 mg/kg), and (or) rapamycin (1 mg/kg) intraperitoneally. Inhibition of mTOR by rapamycin blocked phosphorylated form of ribosomal protein S6, NF-κB p65 activity by increasing degradation of IκB-α in parallel with HIF-1α expression increased by LPS in the kidney, heart, lung, and brain tissues. Rapamycin attenuated the increment in the expression of tumor necrosis factor-α and interleukin-1ß, the inducible nitric oxide synthase, gp91phox, and p47phox in addition to nitrite levels elicited by LPS in tissues or sera. Concomitantly, rapamycin treatment reduced microglial activation, brain expression of caspase-3, and Bcl-2-associated X protein while it increased expression of B cell lymphoma 2 induced by LPS. Overall, this study supports the hypothesis that mTOR contributes to the detrimental effect of LPS-induced systemic inflammatory response associated with neuroinflammation via IκB-α/NF-κB/HIF-1α signaling pathway.

Brain Testosterone-CYP1B1 (Cytochrome P450 1B1) Generated Metabolite 6ß-Hydroxytestosterone Promotes Neurogenic Hypertension and Inflammation.

Previously, we showed that peripheral administration of 6ß-hydroxytestosterone, a CYP1B1 (cytochrome P450 1B1)-generated metabolite of testosterone, promotes angiotensin II-induced hypertension in male mice. However, the site of action and the underlying mechanism by which 6ß-hydroxytestosterone contributes to angiotensin II-induced hypertension is not known. Angiotensin II increases blood pressure by its central action, and CYP1B1 is expressed in the brain. This study was conducted to determine whether testosterone-CYP1B1 generated metabolite 6ß-hydroxytestosterone locally in the brain promotes the effect of systemic angiotensin II to produce hypertension in male mice. Central CYP1B1 knockdown in wild-type (Cyp1b1+/+) mice by intracerebroventricular-adenovirus-GFP (green fluorescence protein)-CYP1B1-short hairpin (sh)RNA attenuated, whereas reconstitution of CYP1B1 by adenovirus-GFP-CYP1B1-DNA in the paraventricular nucleus but not in subfornical organ in Cyp1b1-/- mice restored angiotensin II-induced increase in systolic blood pressure measured by tail-cuff. Intracerebroventricular-testosterone in orchidectomized (Orchi)-Cyp1b1+/+ but not in Orchi-Cyp1b1-/-, and intracerebroventricular-6ß-hydroxytestosterone in the Orchi-Cyp1b1-/- mice restored the angiotensin II-induced: (1) increase in mean arterial pressure measured by radiotelemetry, and autonomic imbalance; (2) reactive oxygen species production in the subfornical organ and paraventricular nucleus; (3) activation of microglia and astrocyte, and neuroinflammation in the paraventricular nucleus. The effect of intracerebroventricular-6ß-hydroxytestosterone to restore the angiotensin II-induced increase in mean arterial pressure and autonomic imbalance in Orchi-Cyp1b1-/- mice was inhibited by intracerebroventricular-small interfering (si)RNA-androgen receptor (AR) and GPRC6A (G protein-coupled receptor C6A). These data suggest that testosterone-CYP1B1-generated metabolite 6ß-hydroxytestosterone, most likely in the paraventricular nucleus via AR and GPRC6A, contributes to angiotensin II-induced hypertension and neuroinflammation in male mice.

Central CYP1B1 (Cytochrome P450 1B1)-Estradiol Metabolite 2-Methoxyestradiol Protects From Hypertension and Neuroinflammation in Female Mice.

Previously, we showed that peripheral administration of 2-ME (2-methoxyestradiol), a CYP1B1 (cytochrome P450 1B1)-catechol-O-methyltransferase (COMT) generated metabolite of E2 (17ß-Estradiol), protects against angiotensin II-induced hypertension in female mice. The demonstration that central E2 inhibits angiotensin II-induced hypertension, together with the expression of CYP1B1 in the brain, led us to hypothesize that E2-CYP1B1 generated metabolite 2-ME in the brain mediates its protective action against angiotensin II-induced hypertension in female mice. To test this hypothesis, we examined the effect of intracerebroventricularly (ICV) administered E2 in ovariectomized (OVX)-wild-type (Cyp1b1+/+) and OVX-Cyp1b1-/- mice on the action of systemic angiotensin II. ICV-E2 attenuated the angiotensin II-induced increase in mean arterial blood pressure, impairment of baroreflex sensitivity, and sympathetic activity in OVX-Cyp1b1+/+ but not in ICV-injected short interfering (si)RNA-COMT or OVX-Cyp1b1-/- mice. ICV-2-ME attenuated the angiotensin II-induced increase in blood pressure in OVX-Cyp1b1-/- mice; this effect was inhibited by ICV-siRNA estrogen receptor-α (ERα) and G protein-coupled estrogen receptor 1 (GPER1). ICV-E2 in OVX-Cyp1b1+/+ but not in OVX-Cyp1b1-/- mice and 2-ME in the OVX-Cyp1b1-/- inhibited angiotensin II-induced increase in reactive oxygen species production in the subfornical organ and paraventricular nucleus, activation of microglia and astrocyte, and neuroinflammation in paraventricular nucleus. Furthermore, central CYP1B1 gene disruption in Cyp1b1+/+ mice by ICV-adenovirus-GFP (green fluorescence protein)-CYP1B1-short hairpin (sh)RNA elevated, while reconstitution by adenovirus-GFP-CYP1B1-DNA in the paraventricular nucleus but not in subfornical organ in Cyp1b1-/- mice attenuated the angiotensin II-induced increase in systolic blood pressure. These data suggest that E2-CYP1B1-COMT generated metabolite 2-ME, most likely in the paraventricular nucleus via estrogen receptor-α and GPER1, protects against angiotensin II-induced hypertension and neuroinflammation in female mice.

6ß-Hydroxytestosterone, a metabolite of testosterone generated by CYP1B1, contributes to vascular changes in angiotensin II-induced hypertension in male mice.

BACKGROUND: Previously, we showed that 6ß-hydroxytestosterone (6ß-OHT), a cytochrome P450 1B1 (CYP1B1)-derived metabolite of testosterone, contributes to angiotensin II (Ang II)-induced hypertension in male mice. This study was conducted to test the hypothesis that 6ß-OHT contributes to increased vascular reactivity, endothelial dysfunction, vascular hypertrophy, and reactive oxygen species production associated with Ang II-induced hypertension. METHODS: Eight- to 10-week-old intact or castrated C57BL/6 J (Cyp1b1+/+ and Cyp1b1-/-) mice were anesthetized for implantation of a micro-osmotic pump which delivered Ang II (700 ng/kg/day) or saline for 14 days. Mice were injected with 6ß-OHT (15 µg/g b.w every third day), flutamide (8 mg/kg every day), or its vehicle. Blood pressure was measured via tail-cuff. Vascular reactivity, endothelial-dependent and endothelial-independent vasodilation, media to lumen ratio, fibrosis by collagen deposition, and reactive oxygen species production by dihydroethidium staining were determined in the isolated thoracic aorta. RESULTS: The response of thoracic aorta to phenylephrine and endothelin-1 was increased in Ang II-infused Cyp1b1+/+ mice compared to intact Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice; these effects of Ang II were restored by treatment with 6ß-OHT. Ang II infusion caused endothelial dysfunction, as indicated by decreased relaxation of the aorta to acetylcholine in Cyp1b1+/+ but not Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice. 6ß-OHT did not alter Ang II-induced endothelial dysfunction in Cyp1b1+/+ mice but restored it in Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice. Ang II infusion increased media to lumen ratio and caused fibrosis and reactive oxygen species production in the aorta of Cyp1b1+/+ mice. These effects were minimized in the aorta of Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice and restored by treatment with 6ß-OHT. Treatment with the androgen receptor antagonist flutamide reduced blood pressure and vascular hypertrophy in castrated Ang II-infused mice injected with 6ß-OHT. CONCLUSIONS: 6ß-OHT is required for the action of Ang II to increase vascular reactivity and cause endothelial dysfunction, hypertrophy, and increase in oxygen radical production. The effect of 6ß-OHT in mediating Ang II-induced hypertension and associated hypertrophy is dependent on the androgen receptor. Therefore, CYP1B1 could serve as a novel target for the development of therapeutics to treat vascular changes in hypertensive males.

Eicosanoids derived from cytochrome P450 pathway of arachidonic acid and inflammatory shock.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Septic shock, the most common form of vasodilatory shock, is a subset of sepsis in which circulatory and cellular/metabolic abnormalities are severe enough to increase mortality. Inflammatory shock constitutes the hallmark of sepsis, but also a final common pathway of any form of severe long-term tissue hypoperfusion. The pathogenesis of inflammatory shock seems to be due to circulating substances released by pathogens (e.g., bacterial endotoxins) and host immuno-inflammatory responses (e.g., changes in the production of histamine, bradykinin, serotonin, nitric oxide [NO], reactive nitrogen and oxygen species, and arachidonic acid [AA]-derived eicosanoids mainly through NO synthase, cyclooxygenase, and cytochrome P450 [CYP] pathways, and proinflammatory cytokine formation). Therefore, refractory hypotension to vasoconstrictors with end-organ hypoperfusion is a life threatening feature of inflammatory shock. This review summarizes the current knowledge regarding the role of eicosanoids derived from CYP pathway of AA in animal models of inflammatory shock syndromes with an emphasis on septic shock in addition to potential therapeutic strategies targeting specific CYP isoforms responsible for proinflammatory/anti-inflammatory mediator production.

Modulation of oxidative-nitrosative stress and inflammatory response by rapamycin in target and distant organs in rats exposed to hindlimb ischemia-reperfusion: the role of mammalian target of rapamycin.

Mammalian target of rapamycin (mTOR) has been recognized with potential immunomodulatory properties playing an important role in various physiopathological processes including ischemia-reperfusion (I/R) injury. I/R injury stimulate reactive oxygen and nitrogen species by activating nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase, respectively. Controversial results have been obtained in different I/R models following localized I/R; however, the precise role of the mTOR signaling pathway remains undefined. The objective of the current study was to evaluate the role of the mTOR in oxidative-nitrosative stress and inflammation in hindlimb I/R-induced injury in target and remote organ injuries. In rats subjected to I/R, an increased expression of ribosomal protein S6 (rpS6), inhibitor κB (IκB)-α, nuclear factor-κB (NF-κB) p65, inducible nitric oxide synthase, cyclooxygenase 2, gp91phox, and levels of tumor necrosis factor α, nitrite, nitrotyrosine, malondialdehyde and the activities of myeloperoxidase and catalase in the tissues and (or) sera were detected. Treatment with rapamycin, a selective inhibitor of mTOR, reversed all the I/R-induced changes as manifested by its anti-inflammatory and antioxidant effects in kidney and gastrocnemius muscle of rats. Collectively, these findings suggest that rapamycin protects against I/R-induced oxidative-nitrosative stress and inflammation leading to organ injuries via suppression of mTOR/IκB-α/NF-κB signaling pathway.

NF-κB activation mediates LPS-or zymosan-induced hypotension and inflammation reversed by BAY61-3606, a selective Syk inhibitor, in rat models of septic and non-septic shock.

We have previously demonstrated that the activation of the spleen tyrosine kinase (Syk)/inhibitory-κB (IκB)-α/nuclear factor-κB (NF-κB) p65 signalling pathway contributes to hypotension and inflammatory response in a rat models of zymosan (ZYM)-induced non-septic shock. The purpose of this study was to further examine the possible mechanism underlying the effect of inhibition of Syk by BAY61-3606 via NF-κB activity at the level of nuclear translocation regarding the production of vasodilator and proinflammatory mediators in lipopolysaccharide (LPS) (septic)- and ZYM (non-septic)-induced shock. Administration of LPS (10 mg/kg, ip) or ZYM (500 mg/kg, ip) to male Wistar rats decreased mean arterial pressure and increased heart rate that was associated with an increase in the activities of cyclooxygenase and nitric oxide synthase, tumour necrosis factor-α, and interleukin-8 levels, and NF-κB activation and nuclear translocation in sera and/or cardiovascular and renal tissues. BAY61-3606 (3 mg/kg, ip), the selective Syk inhibitor, given 1 hour after LPS- or ZYM injection reversed all the above-mentioned effects. These results suggest that Syk contributes to the LPS- or ZYM-induced hypotension and inflammation associated with transactivation of NF-κB in septic and non-septic shock.

Cytochrome P450 1B1 Is Critical for Neointimal Growth in Wire-Injured Carotid Artery of Male Mice.

Background We have reported that cytochrome P450 1B1 ( CYP 1B1), expressed in cardiovascular tissues, contributes to angiotensin II -induced vascular smooth muscle cell ( VSMC ) migration and proliferation and development of hypertension in various experimental animal models via generation of reactive oxygen species. This study was conducted to determine the contribution of CYP 1B1 to platelet-derived growth factor-BB-induced VSMC migration and proliferation in vitro and to neointimal growth in vivo. Methods and Results VSMC s isolated from aortas of male Cyp1b1 +/+ and Cyp1b1 -/- mice were used for in vitro experiments. Moreover, carotid arteries of Cyp1b1 +/+ and Cyp1b1 -/- mice were injured with a metal wire to assess neointimal growth after 14 days. Platelet-derived growth factor- BB -induced migration and proliferation and H2O2 production were found to be attenuated in VSMC s from Cyp1b1 -/- mice and in VSMC s of Cyp1b1 +/+ mice treated with 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, a superoxide dismutase and catalase mimetic. In addition, wire injury resulted in neointimal growth, as indicated by increased intimal area, intima/media ratio, and percentage area of restenosis, as well as elastin disorganization and adventitial collagen deposition in carotid arteries of Cyp1b1 +/+ mice, which were minimized in Cyp1b1 -/- mice. Wire injury also increased infiltration of inflammatory and immune cells, as indicated by expression of CD 68+ macrophages and CD 3+ T cells, respectively, in the injured arteries of Cyp1b1 +/+ mice, but not Cyp1b1 -/- mice. Administration of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl attenuated neointimal growth in wire-injured carotid arteries of Cyp1b1 +/+ mice. Conclusions These data suggest that CYP 1B1-dependent oxidative stress contributes to the neointimal growth caused by wire injury of carotid arteries of male mice.

Deletion of DGCR8 in VSMCs of adult mice results in loss of vascular reactivity, reduced blood pressure and neointima formation.

DiGeorge syndrome chromosomal region 8 (DGCR8), a double-stranded-RNA-binding protein, participates in the miRNA biogenesis pathway and contributes to miRNA maturation by interacting with the RNAase III enzyme Drosha in cell nuclei. To investigate the role of DGCR8 in vascular smooth muscle cells (VSMCs) at the postnatal stages, we generated tamoxifen-inducible VSMC specific knockout (iKO) mice by crossing DGCR8loxp/loxp with VSMC specific tamoxifen-inducible Cre transgenic mice SMA-Cre-ERT2. DGCR8iKO mice display reduced body weight one month following tamoxifen treatment and died around 3 months. Blood pressure and vascular reactivity were significantly reduced in DGCR8iKO mice compared to control. Furthermore, loss of DGCR8 in VSMCs inhibited cell proliferation, migration and neointima formation. VSMC differentiation marker genes, including SMA and SM22, were downregulated in DGCR8 iKO mice. The majority of miRNAs were downregulated in DGCR8iKO mice. Disruption of the DGCR8-mediated miRNA biogenesis pathway attenuated multiple signaling pathways including ERK1/2 and AKT. Our results demonstrate that the DGCR8-mediated miRNA pathway is required for maintaining blood pressure, vascular reactivity and vascular wall remodeling at the postnatal stages.