Sex-Specific Differences in Endothelial Function Are Driven by Divergent Mitochondrial Ca2+ Handling.

Background Sex-specific differences in vasodilation are mediated in part by differences in cytosolic Ca2+ handling, but how variations in mitochondrial Ca2+ contributes to this effect remains unknown. Here, we investigated the extent to which mitochondrial Ca2+ entry via the MCU (mitochondrial Ca2+ uniporter) drives sex differences in vasoreactivity in resistance arteries. Methods and Results Enhanced vasodilation of mesenteric resistance arteries to acetylcholine (ACh) was reduced to larger extent in female compared with male mice in 2 genetic models of endothelial MCU ablation. Ex vivo Ca2+ imaging of mesenteric arteries with Fura-2AM confirmed higher cytosolic Ca2+ transients triggered by ACh in arteries from female mice versus male mice. MCU inhibition both strongly reduced cytosolic Ca2+ transients and blocked mitochondrial Ca2+ entry. In cultured human aortic endothelial cells, treatment with physiological concentrations of estradiol enhanced cytosolic Ca2+ transients, Ca2+ buffering capacity, and mitochondrial Ca2+ entry in response to ATP or repeat Ca2+ boluses. Further experiments to establish the mechanisms underlying these effects did not reveal significant differences in the expression of MCU subunits, at either the mRNA or protein level. However, estradiol treatment was associated with an increase in mitochondrial mass, mitochondrial fusion, and the mitochondrial membrane potential and reduced mitochondrial superoxide production. Conclusions Our data confirm that mitochondrial function in endothelial cells differs by sex, with female mice having enhanced Ca2+ uptake capacity, and that these differences are attributable to the presence of more mitochondria and a higher mitochondrial membrane potential in female mice rather than differences in composition of the MCU complex.

Dietary Fatty Acid Saturation Modulates Sphingosine-1-Phosphate-Mediated Vascular Function.

Sphingolipids, modified by dietary fatty acids, are integral components of plasma membrane and caveolae that are also vasoactive compounds. We hypothesized that dietary fatty acid saturation affects vasoconstriction to sphingosine-1-phosphate (S1P) through caveolar regulation of rho kinase. Wild type (WT) and caveolin-1-deficient (cav-1 KO) mice which lack vascular caveolae were fed a low-fat diet (LF), 60% high-saturated fat diet (lard, HF), or 60% fat diet with equal amounts of lard and n-3 polyunsaturated menhaden oil (MO). Weight gain of WT on HF and MO diets was similar while markedly blunted in cav-1 KO. Neither high-fat diet affected the expression of cav-1, rho, or rho kinase in arteries from WT. In cav-1 KO, MO increased the vascular expression of rho but had no effect on rho kinase. HF had no effect on rho or rho kinase expression in cav-1 KO. S1P produced a concentration-dependent constriction of gracilis arteries from WT on LF that was reduced with HF and restored to normal with MO. Constriction to S1P was reduced in cav-1 KO and no longer affected by a high-saturated fat diet. Inhibition of rho kinase which reduced constriction to PE independent of diet in arteries from WT and cav-1 KO only reduced constriction to S1P in arteries from WT fed MO. The data suggest that dietary fatty acids modify vascular responses to S1P by a caveolar-dependent mechanism which is enhanced by dietary n-3 polyunsaturated fats.

Incisive dental implant migration into the nasal septum.

We report the clinical case of a female patient who presented to our emergency department due to a septal abscess caused by the displacement of a dental implant into the nasal septum. The patient underwent surgical treatment for endoscopic foreign body excision and septal abscess drainage. Despite the presence of septal cartilage destruction, the L-shaped structure was preserved and no reconstruction was required. Postoperative healing was uneventful.

Dietary fats modify vascular fat composition, eNOS localization within lipid rafts and vascular function in obesity.

We tested whether dietary fatty acids alter membrane composition shifting localization of signaling pathways within caveolae to determine their role in vascular function. Wild type (WT) and caveolin-1-deficient mice (cav-1 KO), required for vascular caveolae formation, were fed low fat (LF), high saturated fat (HF, 60% kcal from lard), or high-fat diet with 50:50 lard and n-3 polyunsaturated fatty acid-enriched menhaden oil (MO). HF and MO increased body weight and fat in WT but had less effect in cav-1 KO. MO increased unsaturated fatty acids and the unsaturation index of aorta from WT and cav-1 KO. In LF WT aorta, endothelial nitric oxide synthase (eNOS) was localized to cav-1-enriched low-density fractions which shifted to actin-enriched high-density fractions with acetylcholine (ACh). HF and MO shifted eNOS to high-density fractions in WT aorta which was not affected by ACh. In cav-1 KO aorta, eNOS was localized in low-density non-caveolar fractions but not shifted by ACh or diet. Inducible NOS and cyclooxygenase 1/2 were not localized in low-density fractions or affected by diet, ACh or genotype. ACh-induced dilation of gracilis arteries from HF WT was similar to dilation in LF but the NOS component was reduced. In WT and cav-1 KO, dilation to ACh was enhanced by MO through increased role for NOS and cyclooxygenase. We conclude that dietary fats affect vascular fatty acid composition and membrane localization of eNOS but the contribution of eNOS and cyclooxygenase in ACh-mediated vascular responses is independent of lipid rafts.

Endothelial CaMKII as a regulator of eNOS activity and NO-mediated vasoreactivity.

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase important in transducing intracellular Ca2+ signals. While in vitro data regarding the role of CaMKII in the regulation of endothelial nitric oxide synthase (eNOS) are contradictory, its role in endothelial function in vivo remains unknown. Using two novel transgenic models to express CaMKII inhibitor peptides selectively in endothelium, we examined the effect of CaMKII on eNOS activation, NO production, vasomotor tone and blood pressure. Under baseline conditions, CaMKII activation was low in the aortic wall. Consistently, systolic and diastolic blood pressure, heart rate and plasma NO levels were unaltered by endothelial CaMKII inhibition. Moreover, endothelial CaMKII inhibition had no significant effect on NO-dependent vasodilation. These results were confirmed in studies of aortic rings transduced with adenovirus expressing a CaMKII inhibitor peptide. In cultured endothelial cells, bradykinin treatment produced the anticipated rapid influx of Ca2+ and transient CaMKII and eNOS activation, whereas CaMKII inhibition blocked eNOS phosphorylation on Ser-1179 and dephosphorylation at Thr-497. Ca2+/CaM binding to eNOS and resultant NO production in vitro were decreased under CaMKII inhibition. Our results demonstrate that CaMKII plays an important role in transient bradykinin-driven eNOS activation in vitro, but does not regulate NO production, vasorelaxation or blood pressure in vivo under baseline conditions.

Role of CaMKII in Ang-II-dependent small artery remodeling.

Angiotensin-II (Ang-II) is a well-established mediator of vascular remodeling. The multifunctional calcium-calmodulin-dependent kinase II (CaMKII) is activated by Ang-II and regulates Erk1/2 and Akt-dependent signaling in cultured smooth muscle cells in vitro. Its role in Ang-II-dependent vascular remodeling in vivo is far less defined. Using a model of transgenic CaMKII inhibition selectively in smooth muscle cells, we found that CaMKII inhibition exaggerated remodeling after chronic Ang-II treatment and agonist-dependent vasoconstriction in second-order mesenteric arteries. These findings were associated with increased mRNA and protein expression of smooth muscle structural proteins. As a potential mechanism, CaMKII reduced serum response factor-dependent transcriptional activity. In summary, our findings identify CaMKII as an important regulator of smooth muscle function in Ang-II hypertension in vivo.

Calcium/calmodulin-dependent kinase II inhibition in smooth muscle reduces angiotensin II-induced hypertension by controlling aortic remodeling and baroreceptor function.

BACKGROUND: Multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMCs), but its function in experimental hypertension has not been explored. The aim of this study was to determine the impact of CaMKII inhibition selectively in VSMCs on Ang II hypertension. METHODS AND RESULTS: Transgenic expression of a CaMKII peptide inhibitor in VSMCs (TG SM-CaMKIIN model) reduced the blood pressure response to chronic Ang II infusion. The aortic depressor nerve activity was reset in hypertensive versus normotensive wild-type animals but not in TG SM-CaMKIIN mice, suggesting that changes in baroreceptor activity account for the blood pressure difference between genotypes. Accordingly, aortic pulse wave velocity, a measure of arterial wall stiffness and a determinant of baroreceptor activity, increased in hypertensive versus normotensive wild-type animals but did not change in TG SM-CaMKIIN mice. Moreover, examination of blood pressure and heart rate under ganglionic blockade revealed that VSMC CaMKII inhibition abolished the augmented efferent sympathetic outflow and renal and splanchnic nerve activity in Ang II hypertension. Consequently, we hypothesized that VSMC CaMKII controls baroreceptor activity by modifying arterial wall remodeling in Ang II hypertension. Gene expression analysis in aortas from normotensive and Ang II-infused mice revealed that TG SM-CaMKIIN aortas were protected from Ang II-induced upregulation of genes that control extracellular matrix production, including collagen. VSMC CaMKII inhibition also strongly altered the expression of muscle contractile genes under Ang II. CONCLUSIONS: CaMKII in VSMCs regulates blood pressure under Ang II hypertension by controlling structural gene expression, wall stiffness, and baroreceptor activity.

Differential control of calcium homeostasis and vascular reactivity by Ca2+/calmodulin-dependent kinase II.

The multifunctional Ca(2+)/calmodulin-dependent kinase II (CaMKII) is activated by vasoconstrictors in vascular smooth muscle cells (VSMC), but its impact on vasoconstriction remains unknown. We hypothesized that CaMKII inhibition in VSMC decreases vasoconstriction. Using novel transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), we investigated the effect of CaMKII inhibition on L-type Ca(2+) channel current (ICa), cytoplasmic and sarcoplasmic reticulum Ca(2+), and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual ICa 50 ms after peak amplitude, indicative of loss of L-type Ca(2+) channel-dependent ICa facilitation. Treatment with angiotensin II or phenylephrine increased the intracellular Ca(2+) concentration in wild-type but not TG SM-CaMKIIN VSMC. The difference in intracellular Ca(2+) concentration was abolished by pretreatment with nifedipine, an L-type Ca(2+) channel antagonist. In TG SM-CaMKIIN VSMC, the total sarcoplasmic reticulum Ca(2+) content was reduced as a result of diminished sarcoplasmic reticulum Ca(2+) ATPase activity via impaired derepression of the sarcoplasmic reticulum Ca(2+) ATPase inhibitor phospholamban. Despite the differences in intracellular Ca(2+) concentration, CaMKII inhibition did not alter myogenic tone or vasoconstriction of mesenteric arteries in response to KCl, angiotensin II, and phenylephrine. However, it increased myosin light chain kinase activity. These data suggest that CaMKII activity maintains intracellular calcium homeostasis but is not required for vasoconstriction of mesenteric arteries.

RhoA localization with caveolin-1 regulates vascular contractions to serotonin.

Vascular smooth muscle contraction occurs following an initial response to an increase in intracellular calcium concentration and a sustained response following increases in the sensitivity of contractile proteins to calcium (calcium sensitization). This latter process is regulated by the rhoA/rho kinase pathway and activated by serotonin. In multiple cell types, signaling molecules compartmentalize within caveolae to regulate their activation. We hypothesized that serotonin differentially compartmentalizes rhoA within caveolar versus noncaveolar lipid rafts to regulate sustained vascular contractions. To test this hypothesis, we measured aortic contractions in response to serotonin in wild-type (WT) and cav-1-deficient mice (cav-1 KO). RhoA-dependent contractions in response to serotonin were markedly augmented in arteries from cav-1 KO mice despite a modest reduction in rhoA expression compared with WT. We found that under basal conditions, rhoA in WT arteries was primarily localized within high-density sucrose gradient fractions but temporally shifted to low-density fractions in response to serotonin. In contrast, rhoA in cav-1 KO arteries was primarily in low-density fractions and shifted to high-density fractions in a similar timeframe as that seen in WT mice. We conclude that localization of rhoA to caveolar versus noncaveolar lipid rafts differentially regulates its activation and contractions to rhoA-dependent agonists with greater activation associated with its localization to noncaveolar rafts. Disruption of rhoA localization within caveolae may contribute to increased activation and enhanced vascular contractions in cardiovascular disease.

Overexpression of the SK3 channel alters vascular remodeling during pregnancy, leading to fetal demise.

The maternal cardiovascular system undergoes hemodynamic changes during pregnancy via angiogenesis and vasodilation to ensure adequate perfusion of the placenta. Improper vascularization at the maternal-fetal interface can cause pregnancy complications and poor fetal outcomes. Recent evidence indicates that small conductance Ca(2+)-activated K(+) channel subtype 3 (SK3) contributes to vascular remodeling during pregnancy, and we hypothesized that abnormal SK3 channel expression would alter the ability of the maternal cardiovascular system to adapt to pregnancy demands and lead to poor fetal outcomes. We investigated this hypothesis using transgenic Kcnn3(tm1Jpad)/Kcnn3(tm1Jpad) (SK3(T/T)) mice that overexpress the channel. Isolated pressurized uterine arteries from nonpregnant transgenic SK3(T/T) mice had larger basal diameters and decreased agonist-induced constriction than those from their wild-type counterparts; however, non-receptor-mediated depolarization remained intact. In addition to vascular changes, heart rates and ejection fraction were increased, whereas end systolic volume was reduced in SK3(T/T) mice compared with their wild-type littermates. Uterine sonography of the fetuses on pregnancy day 14 showed a significant decrease in fetal size in SK3(T/T) compared with wild-type mice; thus, SK3(T/T) mice displayed an intrauterine growth-restricted phenotype. The SK3(T/T) mice showed decreased placental thicknesses and higher incidence of fetal loss, losing over half of their complement of pups by midgestation. These results establish that the SK3 channel contributes to both maternal and fetal outcomes during pregnancy and point to the importance of SK3 channel regulation in maintaining a healthy pregnancy.

The role of rho kinase in sex-dependent vascular dysfunction in type 1 diabetes.

We hypothesized that rho/rho kinase plays a role in sex differences in vascular dysfunction of diabetics. Contractions to serotonin were greater in isolated aortic rings from nondiabetic males versus females and increased further in streptozotocin-induced diabetic males but not females. The increased contractions to serotonin in males were reduced by inhibitors of rho kinase (fasudil, Y27632 and H1152) despite no change in expression of rhoA or rho kinase. Contractions to U46619 were not altered by fasudil or Y27632 or the presence of diabetes. In contrast to acute effects of fasudil, chronic treatment with fasudil increased contractions to serotonin in aorta from both non-diabetic and diabetic males. In summary, serotonin-induced contractions were increased in aorta from diabetic males but not females. Although administration of rho kinase inhibitors acutely decreased contractions to serotonin, long-term treatment with fasudil increased contractions. Long-term fasudil treatment may increase compensatory mechanisms to enhance vasoconstrictions.

Sex-dependent differences in Rho activation contribute to contractile dysfunction in type 2 diabetic mice.

The objective of this study was to determine if mechanisms involved in vascular dysfunction in type 2 diabetes differ with sex. Vascular reactivity, expression, and activation of rhoA and rho kinase were measured in aorta from male and female nondiabetic C57BLKS/J and diabetic BKS.Cg-m(+/+) Lepr(db)/J (db/db) mice, a model of type 2 diabetes. Relaxation to acetylcholine and nitroprusside was similar in aorta from nondiabetic male and female mice. Relaxation to acetylcholine was reduced approximately 50% in both male and female diabetic mice. Although inhibition of rho kinase with H-1152 increased relaxation to acetylcholine and nitroprusside in nondiabetic males, it had no effect on the response in either nondiabetic or diabetic females or diabetic males. Contraction to serotonin was increased similarly in male and female diabetic mice compared with nondiabetic mice and was reduced following inhibition of rho kinase with either fasudil or H-1152. Activation of rhoA and its downstream effector, rho kinase, was greater in aorta from diabetic males compared with nondiabetic males. In contrast, there were no differences in vascular activation of rhoA or rho kinase in diabetic females. The increased activity of rhoA and rho kinase in diabetic mice was not due to a change in protein expression of rhoA or rho kinase (ROCK1 and ROCK2) in vessels from either males or females. Although contractile dysfunction in vessels occurs in both male and female diabetic mice, the dysfunction in diabetic males is dependent upon activation of rhoA and rho kinase. Alternative mechanisms affecting rho kinase activation may be involved in females.

RhoA activation contributes to sex differences in vascular contractions.

OBJECTIVE: Studies have suggested that sex differences in endothelial function in part account for the lower incidence of cardiovascular disease in premenopausal women compared with men. Less is known about the role of smooth muscle. We hypothesized that signaling mechanisms that regulate calcium sensitivity in vascular muscle also play a role in determining sex differences in contractile function. METHODS AND RESULTS: In aorta, concentration-dependent contractions to serotonin were greater in male versus female mice whereas contractions to KCl and U46619 were similar. Nitric oxide or other endothelial-derived factors did not account for the difference in responses to serotonin because inhibition of nitric oxide synthase (NOS) with N(G)-nitro-L-arginine, genetic deficiency of endothelial NOS, and removal of endothelium increased contractions but did not abolish the enhanced contractions in aorta from males. Contractions in aorta from both males and females were abolished by a serotonergic 5HT2A receptor antagonist (ketanserin), however there was no sex difference in 5HT2A receptor expression. Activation of RhoA and Rho-kinase by serotonin was greater in aorta from males compared with females, but this was not related to greater expression of RhoA or Rho-kinase isoforms (ROCK1 and ROCK2). The sex difference in aortic contractions to serotonin was abolished by an inhibitor of Rho-kinase, Y27632. CONCLUSION: We conclude that increased contractions to serotonin in aorta from male mice are attributable to differences in RhoA/Rho-kinase activation in smooth muscle independent of differences in the expression of RhoA or Rho-kinase.

Muscarinic (M) receptors in coronary circulation: gene-targeted mice define the role of M2 and M3 receptors in response to acetylcholine.

OBJECTIVE: Determining the role of specific muscarinic (M) receptor subtypes mediating responses to acetylcholine (ACh) has been limited by the specificity of pharmacological agents. Deletion of the gene for M5 receptors abolished response to ACh in cerebral blood vessels but did not affect dilation of coronary arteries. The goal of this study was to determine the M receptors mediating responses to ACh in coronary circulation using mice deficient in M2 or M3 receptors (M2-/-, M3-/-, respectively). METHODS AND RESULTS: Coronary arteries from respective wild-type, M2-/-, or M3-/- mice were isolated, cannulated, and pressurized. Diameter was measured with video microscopy. After preconstriction with U46619, ACh produced dose-dependent dilation of coronary arteries that was similar in wild-type and M2-/- mice. In contrast, dilation of coronary arteries from M3-/- mice to ACh was reduced by approximately 80% compared with wild type. The residual response to ACh was atropine insensitive. Relaxation of coronary arteries to other stimuli was similar in M2-/- and M3-/- mice. Similar results were obtained in aorta rings. CONCLUSIONS: These findings provide the first direct evidence that relaxation to ACh in coronary circulation is mediated predominantly by activation of M3 receptors.

Abnormal coronary function in mice deficient in alpha1H T-type Ca2+ channels.

Calcium ion (Ca2+) influx through voltage-gated Ca2+ channels is important for the regulation of vascular tone. Activation of L-type Ca2+ channels initiates muscle contraction; however, the role of T-type Ca2+ channels (T-channels) is not clear. We show that mice deficient in the alpha1H T-type Ca2+ channel (alpha(1)3.2-null) have constitutively constricted coronary arterioles and focal myocardial fibrosis. Coronary arteries isolated from alpha(1)3.2-null arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside. Furthermore, acute blockade of T-channels with Ni2+ prevented relaxation of wild-type coronary arteries. Thus, Ca2+ influx through alpha1H T-type Ca2+ channels is essential for normal relaxation of coronary arteries.