Cul3 regulates cyclin E1 protein abundance via a degron located within the N-terminal region of cyclin E.

Cyclin E and its binding partner Cdk2 control the G1/S transition in mammalian cells. Increased levels of cyclin E are found in some cancers. Additionally, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with increased cyclin E-Cdk2 activity and poor clinical prognosis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligases, Cul1 and Cul3. Cul1 ubiquitylates phosphorylated cyclin E, but the mechanism through which Cul3 ubiquitylates cyclin E is poorly understood. In experiments to ascertain how Cul3 mediates cyclin E destruction, we identified a degron on cyclin E that Cul3 targets for ubiquitylation. Recognition of the degron and binding of Cul3 does not require a BTB domain-containing adaptor protein. Additionally, this degron is lacking in N-terminally truncated cyclin E. Our results describe a mechanism whereby N-terminally truncated cyclin E can avoid the Cul3-mediated degradation pathway. This mechanism helps to explain the increased activity that is associated with the truncated cyclin E variants that occurs in some cancers.

Dual gain and loss of cullin 3 function mediates familial hyperkalemic hypertension.

Familial hyperkalemic hypertension is caused by mutations in with-no-lysine kinases (WNKs) or in proteins that mediate their degradation, kelch-like 3 (KLHL3) and cullin 3 (CUL3). Although the mechanisms by which WNK and KLHL3 mutations cause the disease are now clear, the effects of the disease-causing CUL3Δ403-459 mutation remain controversial. Possible mechanisms, including hyperneddylation, altered ubiquitin ligase activity, decreased association with the COP9 signalosome (CSN), and increased association with and degradation of KLHL3 have all been postulated. Here, we systematically evaluated the effects of Cul3Δ403-459 using cultured kidney cells. We first identified that the catalytically active CSN subunit jun activation domain-binding protein-1 (JAB1) does not associate with the deleted Cul3 4-helix bundle domain but instead with the adjacent α/ß1 domain, suggesting that altered protein folding underlies the impaired binding. Inhibition of deneddylation with JAB1 siRNA increased Cul3 neddylation and decreased KLHL3 abundance, similar to the Cul3 mutant. We next determined that KLHL3 degradation has both ubiquitin ligase-dependent and -independent components. Proteasomal KLHL3 degradation was enhanced by Cul3Δ403-459; however, autophagic degradation was also upregulated by this Cul3 mutant. Finally, to evaluate whether deficient substrate adaptor was responsible for the disease, we restored KLHL3 to wild-type (WT) Cul3 levels. In the absence of WT Cul3, WNK4 was not degraded, demonstrating that Cul3Δ403-459 itself cannot degrade WNK4; conversely, when WT Cul3 was present, as in diseased humans, WNK4 degradation was restored. In conclusion, deletion of exon 9 from Cul3 generates a protein that is itself ubiquitin-ligase defective but also capable of enhanced autophagocytic KLHL3 degradation, thereby exerting dominant-negative effects on the WT allele.

Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors.

Cullin-Ring ubiquitin ligases regulate protein turnover by promoting the ubiquitination of substrate proteins, targeting them for proteasomal degradation. It has been shown previously that mutations in Cullin3 (Cul3) causing deletion of 57 amino acids encoded by exon 9 (Cul3Δ9) cause hypertension. Moreover, RhoA activity contributes to vascular constriction and hypertension. We show that ubiquitination and degradation of RhoA is dependent on Cul3 in HEK293T cells in which Cul3 expression is ablated by either siRNA or by CRISPR-Cas9 genome editing. The latter was used to generate a Cul3-null cell line (HEK293T(Cul3KO)). When expressed in these cells, Cul3Δ9 supported reduced ubiquitin ligase activity toward RhoA compared with equivalent levels of wild-type Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3Δ9 to the E3 ubiquitin ligase Rbx1 and neddylation of Cul3Δ9 were impaired significantly compared with Cul3WT. Conversely, Cul3Δ9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3Δ9 also forms unstable dimers with Cul3WT, disrupting dimers of Cul3WT complexes that are required for efficient ubiquitination of some substrates. Indeed, coexpression of Cul3WT and Cul3Δ9 in HEK293T(Cul3KO) cells resulted in a decrease in the active form of Cul3WT. We conclude that Cul3Δ9-associated ubiquitin ligase activity toward RhoA is impaired and suggest that Cul3Δ9 mutations may act dominantly by sequestering substrate adaptors and disrupting Cul3WT complexes.

Role of CYP1A1 in modulating the vascular and blood pressure benefits of omega-3 polyunsaturated fatty acids.

The mechanisms that mediate the cardiovascular protective effects of omega 3 (n-3) polyunsaturated fatty acids (PUFAs) have not been fully elucidated. Cytochrome P450 1A1 efficiently metabolizes n-3 PUFAs to potent vasodilators. Thus, we hypothesized that dietary n-3 PUFAs increase nitric oxide (NO)-dependent blood pressure regulation and vasodilation in a CYP1A1-dependent manner. CYP1A1 wild-type (WT) and knockout (KO) mice were fed an n-3 or n-6 PUFA-enriched diet for 8 weeks and were analyzed for tissue fatty acids and metabolites, NO-dependent blood pressure regulation, NO-dependent vasodilation of acetylcholine (ACh) in mesenteric resistance arterioles, and endothelial NO synthase (eNOS) and phospho-Ser1177-eNOS expression in the aorta. All mice fed the n-3 PUFA diet showed significantly higher levels of n-3 PUFAs and their metabolites, and significantly lower levels of n-6 PUFAs and their metabolites. In addition, KO mice on the n-3 PUFA diet accumulated significantly higher levels of n-3 PUFAs in the aorta and kidney without a parallel increase in the levels of their metabolites. Moreover, KO mice exhibited significantly less NO-dependent regulation of blood pressure on the n-3 PUFA diet and significantly less NO-dependent, ACh-mediated vasodilation in mesenteric arterioles on both diets. Finally, the n-3 PUFA diet significantly increased aortic phospho-Ser1177-eNOS/eNOS ratio in the WT compared with KO mice. These data demonstrate that CYP1A1 contributes to eNOS activation, NO bioavailability, and NO-dependent blood pressure regulation mediated by dietary n-3 PUFAs.

Elevated blood pressure in cytochrome P4501A1 knockout mice is associated with reduced vasodilation to omega-3 polyunsaturated fatty acids.

In vitro cytochrome P4501A1 (CYP1A1) metabolizes omega-3 polyunsaturated fatty acids (n-3 PUFAs); eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), primarily to 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP), respectively. These metabolites have been shown to mediate vasodilation via increases in nitric oxide (NO) and activation of potassium channels. We hypothesized that genetic deletion of CYP1A1 would reduce vasodilatory responses to n-3 PUFAs, but not the metabolites, and increase blood pressure (BP) due to decreases in NO. We assessed BP by radiotelemetry in CYP1A1 wildtype (WT) and knockout (KO) mice±NO synthase (NOS) inhibitor. We also assessed vasodilation to acetylcholine (ACh), EPA, DHA, 17,18-EEQ and 19,20-EDP in aorta and mesenteric arterioles. Further, we assessed vasodilation to an NO donor and to DHA±inhibitors of potassium channels. CYP1A1 KO mice were hypertensive, compared to WT, (mean BP in mmHg, WT 103±1, KO 116±1, n=5/genotype, p<0.05), and exhibited a reduced heart rate (beats per minute, WT 575±5; KO 530±7; p<0.05). However, BP responses to NOS inhibition and vasorelaxation responses to ACh and an NO donor were normal in CYP1A1 KO mice, suggesting that NO bioavailability was not reduced. In contrast, CYP1A1 KO mice exhibited significantly attenuated vasorelaxation responses to EPA and DHA in both the aorta and mesenteric arterioles, but normal vasorelaxation responses to the CYP1A1 metabolites, 17,18-EEQ and 19,20-EDP, and normal responses to potassium channel inhibition. Taken together these data suggest that CYP1A1 metabolizes n-3 PUFAs to vasodilators in vivo and the loss of these vasodilators may lead to increases in BP.

Endothelial Cullin3 Mutation Impairs Nitric Oxide-Mediated Vasodilation and Promotes Salt-Induced Hypertension.

Human hypertension caused by in-frame deletion of CULLIN3 exon-9 (Cul3∆9) is driven by renal and vascular mechanisms. We bred conditionally activatable Cul3∆9 transgenic mice with tamoxifen-inducible Tie2-CREERT2 mice to test the importance of endothelial Cul3. The resultant mice (E-Cul3∆9) trended towards elevated nighttime blood pressure (BP) correlated with increased nighttime activity, but displayed no difference in daytime BP or activity. Male and female E-Cul3∆9 mice together exhibited a decline in endothelial-dependent relaxation in carotid artery. Male but not female E-Cul3∆9 mice displayed severe endothelial dysfunction in cerebral basilar artery. There was no impairment in mesenteric artery and no difference in smooth muscle function, suggesting the effects of Cul3∆9 are arterial bed-specific and sex-dependent. Expression of Cul3∆9 in primary mouse aortic endothelial cells decreased endogenous Cul3 protein, phosphorylated (S1177) endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Cul3∆9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor okadaic acid, but not by a PP1 inhibitor tautomycetin. Because NO deficiency contributes to salt-induced hypertension, we tested the salt-sensitivity of E-Cul3∆9 mice. While both male and female E-Cul3∆9 mice developed salt-induced hypertension and renal injury, the pressor effect of salt was greater in female mutants. The increased salt-sensitivity in female E-Cul3∆9 mice was associated with decreased renovascular relaxation and impaired natriuresis in response to a sodium load. Thus, CUL3 mutations in the endothelium may contribute to human hypertension in part through decreased endothelial NO bioavailability, renovascular dysfunction, and increased salt-sensitivity of BP.

Failure to vasodilate in response to salt loading blunts renal blood flow and causes salt-sensitive hypertension.

AIMS: Salt-sensitive (SS) hypertension is accompanied by impaired vasodilation in the systemic and renal circulation. However, the causal relationship between vascular dysfunction and salt-induced hypertension remains controversial. We sought to determine whether primary vascular dysfunction, characterized by a failure to vasodilate during salt loading, plays a causal role in the pathogenesis of SS hypertension. METHODS AND RESULTS: Mice selectively expressing a peroxisome proliferator-activated receptor γ dominant-negative mutation in vascular smooth muscle (S-P467L) exhibited progressive SS hypertension during a 4 week high salt diet (HSD). This was associated with severely impaired vasodilation in systemic and renal vessels. Salt-induced impairment of vasodilation occurred as early as 3 days after HSD, which preceded the onset of SS hypertension. Notably, the overt salt-induced hypertension in S-P467L mice was not driven by higher cardiac output, implying elevations in peripheral vascular resistance. In keeping with this, HSD-fed S-P467L mice exhibited decreased smooth muscle responsiveness to nitric oxide (NO) in systemic vessels. HSD-fed S-P467L mice also exhibited elevated albuminuria and a blunted increase in urinary NO metabolites which was associated with blunted renal blood flow and increased sodium retention mediated by a lack of HSD-induced suppression of NKCC2. Blocking NKCC2 function prevented the salt-induced increase in blood pressure in S-P467L mice. CONCLUSION: We conclude that failure to vasodilate in response to salt loading causes SS hypertension by restricting renal perfusion and reducing renal NO through a mechanism involving NKCC2 in a mouse model of vascular peroxisome proliferator-activated receptor γ impairment.

Increased Susceptibility of Mice Lacking Renin-b to Angiotensin II-Induced Organ Damage.

Several cardiac and renal diseases are attributed to a dysregulation of the renin-angiotensin system. Renin, the rate-limiting enzyme of the renin-angiotensin system, has 2 isoforms. The classical renin isoform (renin-a) encoding preprorenin is mainly confined to the juxtaglomerular cells and released into the circulation upon stimulation. Alternatively, renin-b is predicted to remain intracellular and is expressed in the brain, heart, and adrenal gland. In the brain, ablation of renin-b (Ren-bNull mice) results in increased brain renin-angiotensin system activity. However, the consequences of renin-b ablation in tissues outside the brain remain unknown. Therefore, we hypothesized that renin-b protects from hypertensive cardiac and renal end-organ damage in mice. Ren-bNull mice exhibited normal blood pressure at baseline. Thus, we induced hypertension by using a slow pressor dose of Ang II (angiotensin II). Ang II increased blood pressure in both wild type and Ren-bNull to the same degree. Although the blood pressure between Ren-bNull and wild-type mice was elevated equally, 4-week infusion of Ang II resulted in exacerbated cardiac remodeling in Ren-bNull mice compared with wild type. Ren-bNull mice also exhibited a modest increase in renal glomerular matrix deposition, elevated plasma aldosterone, and a modestly enhanced dipsogenic response to Ang II. Interestingly, ablation of renin-b strongly suppressed plasma renin, but renal cortical renin mRNA was preserved. Altogether, these data indicate that renin-b might play a protective role in the heart, and thus renin-b could be a potential target to treat hypertensive heart disease.

Conditional deletion of smooth muscle Cullin-3 causes severe progressive hypertension.

Patients with mutations in Cullin-3 (CUL3) exhibit severe early onset hypertension but the contribution of the smooth muscle remains unclear. Conditional genetic ablation of CUL3 in vascular smooth muscle (S-CUL3KO) causes progressive impairment in responsiveness to nitric oxide (NO), rapid development of severe hypertension, and increased arterial stiffness. Loss of CUL3 in primary aortic smooth muscle cells or aorta resulted in decreased expression of the NO receptor, soluble guanylate cyclase (sGC), causing a marked reduction in cGMP production and impaired vasodilation to cGMP analogues. Vasodilation responses to a selective large conductance Ca2+-activated K+-channel activator were normal suggesting that downstream signals which promote smooth muscle-dependent relaxation remained intact. We conclude that smooth muscle specific CUL3 ablation impairs both cGMP production and cGMP responses and that loss of CUL3 function selectively in smooth muscle is sufficient to cause severe hypertension by interfering with the NO-sGC-cGMP pathway. Our study provides compelling evidence for the sufficiency of vascular smooth muscle CUL3 as a major regulator of BP. CUL3 mutations cause severe vascular dysfunction, arterial stiffness and hypertension due to defects in vascular smooth muscle.

RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity.

Mice selectively expressing PPARγ dominant negative mutation in vascular smooth muscle exhibit RhoBTB1-deficiency and hypertension. Our rationale was to employ genetic complementation to uncover the mechanism of action of RhoBTB1 in vascular smooth muscle. Inducible smooth muscle-specific restoration of RhoBTB1 fully corrected the hypertension and arterial stiffness by improving vasodilator function. Notably, the cardiovascular protection occurred despite preservation of increased agonist-mediated contraction and RhoA/Rho kinase activity, suggesting RhoBTB1 selectively controls vasodilation. RhoBTB1 augmented the cGMP response to nitric oxide by restraining the activity of phosphodiesterase 5 (PDE5) by acting as a substrate adaptor delivering PDE5 to the Cullin-3 E3 Ring ubiquitin ligase complex for ubiquitination inhibiting PDE5. Angiotensin-II infusion also caused RhoBTB1-deficiency and hypertension which was prevented by smooth muscle specific RhoBTB1 restoration. We conclude that RhoBTB1 protected from hypertension, vascular smooth muscle dysfunction, and arterial stiffness in at least two models of hypertension.

Endothelial PPARγ (Peroxisome Proliferator-Activated Receptor-γ) Protects From Angiotensin II-Induced Endothelial Dysfunction in Adult Offspring Born From Pregnancies Complicated by Hypertension.

Preeclampsia is a hypertensive disorder of pregnancy associated with vascular dysfunction and cardiovascular risk to offspring. We hypothesize that endothelial PPARγ (peroxisome proliferator-activated receptor-γ) provides cardiovascular protection in offspring from pregnancies complicated by hypertension. C57BL/6J dams were bred with E-V290M sires, which express a dominant-negative allele of PPARγ selectively in the endothelium. Arginine vasopressin was infused throughout gestation. Vasopressin elevated maternal blood pressure at gestational day 14 to 15 and urinary protein at day 17 consistent. Systolic blood pressure and vasodilation responses to acetylcholine were similar in vasopressin-exposed offspring compared to offspring from control pregnancies. We treated offspring with a subpressor dose of angiotensin II to test if hypertension during pregnancy predisposes offspring to hypertension. Male and female angiotensin II-treated E-V290M offspring from vasopressin-exposed but not control pregnancy exhibited significant impairment in acetylcholine-induced relaxation in carotid artery. Endothelial dysfunction in angiotensin II-treated E-V290M vasopressin-exposed offspring was attenuated by tempol, an effect which was more prominent in male offspring. Nrf2 (nuclear factor-E2-related factor) protein levels were significantly elevated in aorta from male E-V290M offspring, but not female offspring compared to controls. Blockade of ROCK (Rho-kinase) signaling and incubation with a ROCK2-specific inhibitor improved endothelial function in both male and female E-V290M offspring from vasopressin-exposed pregnancy. Our data suggest that interference with endothelial PPARγ in offspring from vasopressin-exposed pregnancies increases the risk for endothelial dysfunction on exposure to a cardiovascular stressor in adulthood. This implies that endothelial PPARγ provides protection to cardiovascular stressors in offspring of a pregnancy complicated by hypertension and perhaps in preeclampsia.

Interference With Endothelial PPAR (Peroxisome Proliferator-Activated Receptor)-γ Causes Accelerated Cerebral Vascular Dysfunction in Response to Endogenous Renin-Angiotensin System Activation.

Low-salt diet is beneficial in salt-sensitive hypertension but may provoke cardiovascular risk in patients with heart failure, diabetes mellitus, or other cardiovascular abnormalities because of endogenous renin-angiotensin system activation. PPAR (peroxisome proliferator-activated receptor)-γ is a transcription factor which promotes an antioxidant pathway in the endothelium. We studied transgenic mice expressing a dominant-negative mutation in PPAR-γ selectively in the endothelium (E-V290M) to test the hypothesis that endothelial PPAR-γ plays a protective role in response to low salt-mediated renin-angiotensin system activation. Plasma renin and Ang II (angiotensin II) were significantly and equally increased in all mice fed low salt for 6 weeks. Vasorelaxation to acetylcholine was not affected in basilar artery from E-V290M at baseline but was significantly and selectively impaired in E-V290M after low salt. Unlike basilar artery, low salt was not sufficient to induce vascular dysfunction in carotid artery or aorta. Endothelial dysfunction in the basilar artery from E-V290M mice fed low salt was attenuated by scavengers of superoxide, inhibitors of NADPH oxidase, or blockade of the Ang II AT1 (angiotensin type-1) receptor. Simultaneous AT1 and AT2 receptor blockade revealed that the restoration of endothelial function after AT1 receptor blockade was not a consequence of AT2 receptor activation. We conclude that interference with PPAR-γ in the endothelium produces endothelial dysfunction in the cerebral circulation in response to low salt-mediated activation of the endogenous renin-angiotensin system, mediated at least in part, through AT1 receptor activation and perturbed redox homeostasis. Moreover, our data suggest that the cerebral circulation may be particularly sensitive to inhibition of PPAR-γ activity and renin-angiotensin system activation.

Arginine vasopressin infusion is sufficient to model clinical features of preeclampsia in mice.

Copeptin, a marker of arginine vasopressin (AVP) secretion, is elevated throughout human pregnancies complicated by preeclampsia (PE), and AVP infusion throughout gestation is sufficient to induce the major phenotypes of PE in mice. Thus, we hypothesized a role for AVP in the pathogenesis of PE. AVP infusion into pregnant C57BL/6J mice resulted in hypertension, renal glomerular endotheliosis, intrauterine growth restriction, decreased placental growth factor (PGF), altered placental morphology, placental oxidative stress, and placental gene expression consistent with human PE. Interestingly, these changes occurred despite a lack of placental hypoxia or elevations in placental fms-like tyrosine kinase-1 (FLT1). Coinfusion of AVP receptor antagonists and time-restricted infusion of AVP uncovered a mid-gestational role for the AVPR1A receptor in the observed renal pathologies, versus mid- and late-gestational roles for the AVPR2 receptor in the blood pressure and fetal phenotypes. These findings demonstrate that AVP is sufficient to initiate phenotypes of PE in the absence of placental hypoxia, and indicate that AVP may mechanistically (independently, and possibly synergistically with hypoxia) contribute to the development of clinical signs of PE in specific subtypes of human PE. Additionally, they identify divergent and gestational time-specific signaling mechanisms that mediate the development of PE phenotypes in response to AVP.

Mutant Cullin 3 causes familial hyperkalemic hypertension via dominant effects.

Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine [K] Kinase 4 (WNK4), with excessive activation of the downstream Sterile 20 (STE20)/SPS-1-related proline/alanine-rich kinase (SPAK) increasing phosphorylation of the Na+-Cl- cotransporter (NCC). CUL3-Δ9 promotes its own degradation via autoubiquitination, leading to the hypothesis that Cul3 haploinsufficiency causes FHHt. To directly test this, we generated Cul3 heterozygous mice (CUL3-Het), and Cul3 heterozygotes also expressing CUL3-Δ9 (CUL3-Het/Δ9), using an inducible renal epithelial-specific system. Endogenous CUL3 was reduced to 50% in both models, and consistent with autoubiquitination, CUL3-Δ9 protein was undetectable in CUL3-Het/Δ9 kidneys unless primary renal epithelia cells were cultured. Abundances of WNK4 and phosphorylated NCC did not differ between control and CUL3-Het mice, but they were elevated in CUL3-Het/Δ9 mice, which also displayed higher plasma [K+] and blood pressure. Abundance of phosphorylated Na+-K+-2Cl- cotransporter (NKCC2) was also increased, which may contribute to the severity of CUL3-Δ9-mediated FHHt. WNK4 and SPAK localized to puncta in NCC-positive segments but not in NKCC2-positive segments, suggesting differential effects of CUL3-Δ9. These results indicate that Cul3 haploinsufficiency does not cause FHHt, but dominant effects of CUL3-Δ9 are required.

Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanism.

Cullin-3 (CUL3) mutations (CUL3Δ9) were previously identified in hypertensive patients with pseudohypoaldosteronism type-II (PHAII), but the mechanism causing hypertension and whether this is driven by renal tubular or extratubular mechanisms remains unknown. We report that selective expression of CUL3Δ9 in smooth muscle acts by interfering with expression and function of endogenous CUL3, resulting in impaired turnover of the CUL3 substrate RhoA, increased RhoA activity, and augmented RhoA/Rho kinase signaling. This caused vascular dysfunction and increased arterial pressure under baseline conditions and a marked increase in arterial pressure, collagen deposition, and vascular stiffness in response to a subpressor dose of angiotensin II, which did not cause hypertension in control mice. Inhibition of total cullin activity increased the level of CUL3 substrates cyclin E and RhoA, and expression of CUL3Δ9 decreased the level of the active form of endogenous CUL3 in human aortic smooth muscle cells. These data indicate that selective expression of the Cul3Δ9 mutation in vascular smooth muscle phenocopies the hypertension observed in Cul3Δ9 human subjects and suggest that mutations in CUL3 cause human hypertension in part through a mechanism involving smooth muscle dysfunction initiated by a loss of CUL3-mediated degradation of RhoA.

Endothelial cell-specific aryl hydrocarbon receptor knockout mice exhibit hypotension mediated, in part, by an attenuated angiotensin II responsiveness.

Hypotension in aryl hydrocarbon receptor knockout mice (ahr(-/-)) is mediated, in part, by a reduced contribution of angiotensin (Ang) II to basal blood pressure (BP). Since AHR is highly expressed in endothelial cells (EC), we hypothesized that EC-specific ahr(-/-) (ECahr(-/-)) mice would exhibit a similar phenotype. We generated ECahr(-/-) mice by crossing AHR floxed mice (ahr(fx/fx)) to mice expressing Cre recombinase driven by an EC-specific promoter. BP was assessed by radiotelemetry prior to and following an acute injection of Ang II or chronic treatment with an angiotensin converting enzyme inhibitor (ACEi). ECahr(-/-) mice were hypotensive (ECahr(+/+): 116.1±1.4; ECahr(-/-): 107.4±2.0 mmHg, n=11, p<0.05) and exhibited significantly different responses to Ang II and ACEi. While Ang II increased BP in both genotypes, the increase was sustained in ECahr(+/+), whereas the increase in ECahr(-/-) mice steadily declined. Area under the curve analysis showed that Ang II-induced increase in diastolic BP (DBP) over 30 min was significantly lower in ECahr(-/-) mice (ECahr(+/+) 1297±223 mmHg/30 min; ECahr(-/-)(AUC): 504±138 mmHg/30 min, p<0.05). In contrast, while ACEi decreased BP in both genotypes, the subsequent rise in DBP after treatment was significantly delayed in the ECahr(-/-) mice. ECahr(-/-) mice also exhibited reduced vascular and adipose Ang II type 1 receptor (AT1R) expression, and reduced aortic Ang II-dependent vasoconstriction in the presence of vascular adipose. Taken together these data suggest that hypotension in ECahr(-/-) mice results from reduced vascular responsiveness to Ang II that is influenced by AT1R expression and adipose.

Cytochrome P4501A1 is required for vascular dysfunction and hypertension induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

National Health and Nutrition Examination Survey data show an association between hypertension and exposure to dioxin-like halogenated aromatic hydrocarbons (HAHs). Furthermore, chronic exposure of mice to the prototypical HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces reactive oxygen species (ROS), endothelial dysfunction, and hypertension. Because TCDD induces cytochrome P4501A1 (CYP1A1) and CYP1A1 can increase ROS, we tested the hypothesis that TCDD-induced endothelial dysfunction and hypertension are mediated by CYP1A1. CYP1A1 wild-type (WT) and knockout (KO) mice were fed one control or TCDD-containing pill (180 ng TCDD/kg, 5 days/week) for 35 days (n = 10-14/genotype/treatment). Blood pressure was monitored by radiotelemetry, and liver TCDD concentration, CYP1A1 induction, ROS, and aortic reactivity were measured at 35 days. TCDD accumulated to similar levels in livers of both genotypes. TCDD induced CYP1A1 in endothelium of aorta and mesentery without detectable expression in the vessel wall. TCDD also induced superoxide anion production, measured by NADPH-dependent lucigenin luminescence, in aorta, heart, and kidney of CYP1A1 WT mice but not KO mice. In contrast, TCDD induced hydrogen peroxide, measured by amplex red assay, to similar levels in aorta of CYP1A1 WT and KO mice but not in heart or kidney. TCDD reduced acetylcholine-dependent vasorelaxation in aortic rings of CYP1A1 WT mice but not in KO mice. Finally, TCDD steadily increased blood pressure after 15 days, which plateaued after 25 days (+20 mmHg) in CYP1A1 WT mice but failed to alter blood pressure in KO mice. These results demonstrate that CYP1A1 is required for TCDD-induced cardiovascular superoxide anion production, endothelial dysfunction, and hypertension.

An activated renin-angiotensin system maintains normal blood pressure in aryl hydrocarbon receptor heterozygous mice but not in null mice.

It has been postulated that fetal vascular abnormalities in aryl hydrocarbon receptor null (ahr(-/-)) mice may alter cardiovascular homeostasis in adulthood. We tested the hypothesis that blood pressure regulation in adult heterozygous mice (ahr(+/-)) would be normal, compared to ahr(-/-) mice, since no vascular abnormalities have been reported in the heterozygote animals. Mean arterial blood pressure (MAP) was measured using radiotelemetry prior to and during treatment with inhibitors of the autonomic nervous system, nitric oxide synthase (NOS), angiotensin converting enzyme (ACE), or endothelin-1 A receptor (ET(A)). Also, indices of renin-angiotensin system (RAS) activation were measured. ahr(+/-) and ahr(-/-) mice were normotensive and hypotensive, respectively, compared to wild-type (ahr(+/+)) littermates. Responses of all genotypes to autonomic nervous system inhibition were normal. ahr(+/-) mice responded normally to NOS inhibition, while the responses of ahr(-/-) mice were significantly blunted. In contrast, ahr(+/-) mice were significantly more responsive to inhibition of ACE, an ET(A) antagonist, or both, while ahr(-/-) mice were significantly less responsive to ACE inhibition and more responsive to an ET(A) antagonist. ahr(+/-) mice also exhibited significant increases in plasma renin and ACE activity, plasma sodium, and urine osmolality, indicative of RAS activation. Thus, normotension in ahr(+/-) mice appears to be maintained by increased RAS and ET-1 signaling, while hypotension in ahr(-/-) mice may result from decreased RAS signaling. In conclusion, despite the lack of overt fetal vascular abnormalities in ahr(+/-) mice, the loss of a single ahr allele has a significant effect on blood pressure regulation.

Loss of the aryl hydrocarbon receptor induces hypoxemia, endothelin-1, and systemic hypertension at modest altitude.

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix Per-Arnt-Sim transcription factor that mediates induction of metabolic enzymes and toxicity of certain environmental pollutants. Although AHR knockout (KO) mice develop cardiac hypertrophy, conflicting reports associate this pathology with hypotension or endothelin (ET)-1-dependent hypertension. Because hypertension occurred at modest altitude, we tested the hypothesis that loss of AHR increases the sensitivity to hypoxia-induced ET-1, contributing to systemic hypertension. We found that AHR KO mice were hypertensive at modest altitude (1632 m) but hypotensive at low altitude (225 m). When AHR KO mice residing at 1632 m were exposed to the partial pressure of inspired oxygen (PIO(2)) at sea level for 11 days, blood pressure declined to levels measured at 225 m. Although plasma ET-1 in AHR KO mice was significantly elevated at 1632 m and decreased at 225 m and sea level PIO(2), pulmonary prepro-ET-1 mRNA was significantly reduced at 1632 m and decreased further at 225 m and sea level PIO(2). Blood gas analysis revealed that AHR KO mice were hypoxemic, hypercapnic, and acidotic at 1632 m, values that were attenuated and normalized after 24 hours and 11 days under sea level PIO(2), respectively. Lastly, AHR inactivation in endothelial cells by small interfering RNA significantly reduced basal prepro-ET-1 mRNA but did not alter hypoxia-induced expression. Our studies establish the AHR KO mouse as a model in which modest decreases in PIO(2) lead to hypoxemia, increased plasma ET-1, and systemic hypertension without increased pulmonary prepro-ET-1 mRNA expression.