Pimonrat Ketsawatsomkron

Deletion of Protein Tyrosine Phosphatase 1b Improves Peripheral Insulin Resistance and Vascular Function in Obese, Leptin-Resistant Mice via Reduced Oxidant Tone

Rationale: Obesity is a risk factor for cardiovascular dysfunction, yet the underlying factors driving this impaired function remain poorly understood. Insulin resistance is a common pathology in obese patients and has been shown to impair vascular function. Whether insulin resistance or obesity, itself, is causal remains unclear. Objective: The present study tested the hypothesis that insulin resistance is the underlying mediator for impaired NO-mediated dilation in obesity by genetic deletion of the insulin-desensitizing enzyme protein tyrosine phosphatase (PTP)1B in db/db mice. Methods and Results: The db/db mouse is morbidly obese, insulin-resistant, and has tissue-specific elevation in PTP1B expression compared to lean controls. In db/db mice, PTP1B deletion improved glucose clearance, dyslipidemia, and insulin receptor signaling in muscle and fat. Hepatic insulin signaling in db/db mice was not improved by deletion of PTP1B, indicating specific amelioration of peripheral insulin resistance. Additionally, obese mice demonstrate an impaired endothelium dependent and independent vasodilation to acetylcholine and sodium nitroprusside, respectively. This impairment, which correlated with increased superoxide in the db/db mice, was corrected by superoxide scavenging. Increased superoxide production was associated with increased expression of NAD(P)H oxidase 1 and its molecular regulators, Noxo1 and Noxa1. Conclusions: Deletion of PTP1B improved both endothelium dependent and independent NO-mediated dilation and reduced superoxide generation in db/db mice. PTP1B deletion did not affect any vascular function in lean mice. Taken together, these data reveal a role for peripheral insulin resistance as the mediator of vascular dysfunction in obesity.

Impaired insulin-mediated vasorelaxation in a nonobese model of type 2 diabetes: role of endothelin-1.

Insulin resistance involves decreased phosphorylation of insulin receptor substrate (IRS) proteins and (or) Akt. In the vasculature, modulated Akt phosphorylation may cause impaired vasorelaxation via decreased eNOS activation. Diet-induced insulin resistance enhances endothelin-1(ET-1)-mediated vasoconstriction and prevents vasodilatation to insulin. Presently, we evaluated insulin-mediated vascular relaxation, assessed molecular markers of the insulin signaling pathway, and determined the involvement of ET-1 in response to insulin by using selective ETA- or ETB-receptor blockade in a lean model of type 2 diabetes. Dose-response curves to insulin (0.01-100 ng/mL) were generated with wire myograph using thoracic aorta rings from control Wistar or diabetic Goto-Kakizaki (GK) rats (n=3-11). Maximal relaxation (Rmax) to insulin was significantly impaired and insulin sensitivity was decreased in the GK group. Preincubation with 1 micromol/L BQ-123 or BQ-788 for ETA- and ETB-receptor blockade, respectively, resulted in improved insulin sensitivity. Immunoblotting for native and phosphorylated Akt and IRS-1 revealed a decrease in Akt activation in the GK group. In vivo hyperinsulinemic euglycemic clamp studies showed decreased glucose utilization in GK rats, indicative of insulin resistance. These findings provide evidence that vascular insulin resistance occurs in a nonobese model of diabetes and that both ET receptor subtypes are involved in vascular relaxation to insulin.

Differential Control of Calcium Homeostasis and Vascular Reactivity by CaMKII

The multifunctional Ca2+/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 Ca2+ channel current (ICa), cytoplasmic and sarcoplasmic reticulum Ca2+, 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 Ca2+ channel–dependent ICa facilitation. Treatment with angiotensin II or phenylephrine increased the intracellular Ca2+ concentration in wild-type but not TG SM-CaMKIIN VSMC. The difference in intracellular Ca2+ concentration was abolished by pretreatment with nifedipine, an L-type Ca2+ channel antagonist. In TG SM-CaMKIIN VSMC, the total sarcoplasmic reticulum Ca2+ content was reduced as a result of diminished sarcoplasmic reticulum Ca2+ ATPase activity via impaired derepression of the sarcoplasmic reticulum Ca2+ ATPase inhibitor phospholamban. Despite the differences in intracellular Ca2+ 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.

Role of Peroxisome Proliferator–Activated Receptor-γ in Vascular Muscle in the Cerebral Circulation

Although peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) is thought to play a protective role in the vasculature, its cell-specific effect, particularly in resistance vessels, is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPAR&ggr; in vascular muscle would impair NO-dependent responses and augment vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPAR&ggr; (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPAR&ggr; in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses after interference with PPAR&ggr; involved Rho kinase with no apparent contribution by oxidative stress–related mechanisms. These findings support the concept that via effects on Rho kinase–dependent signaling, PPAR&ggr; in vascular muscle is a major determinant of vascular tone in resistance vessels and, in particular, NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow and the pathogenesis of large and small vessel disease in brain.Although peroxisome proliferator-activated receptor-gamma (PPARγ) is thought to play a protective role in the vasculature, its cell-specific impact, particularly in resistance vessels is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPARγ in vascular muscle would impair NO-dependent responses as well as augmenting vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPARγ (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPARγ in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses following interference with PPARγ involved Rho kinase with no apparent contribution by oxidative stress-related mechanisms. These findings support the concept that via effects on Rho kinase-dependent signaling, PPARγ in vascular muscle is a major determinant of vascular tone in resistance vessels, and in particular NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow as well as the pathogenesis of large and small vessel disease in brain.

Genetic Interference With Peroxisome Proliferator–Activated Receptor γ in Smooth Muscle Enhances Myogenic Tone in the Cerebrovasculature via A Rho Kinase–Dependent Mechanism

Myogenic responses by resistance vessels are a key component of autoregulation in brain, thus playing a crucial role in regulating cerebral blood flow and protecting the blood–brain barrier against potentially detrimental elevations in blood pressure. Although cerebrovascular disease is often accompanied by alterations in myogenic responses, mechanisms that control these changes are poorly understood. Peroxisome proliferator–activated receptor &ggr; has emerged as a regulator of vascular tone. We hypothesized that interference with peroxisome proliferator–activated receptor &ggr; in smooth muscle would augment myogenic responses in cerebral arteries. We studied transgenic mice expressing a dominant-negative mutation in peroxisome proliferator–activated receptor &ggr; selectively in smooth muscle (S-P467L) and nontransgenic littermates. Myogenic tone in middle cerebral arteries from S-P467L was elevated 3-fold when compared with nontransgenic littermates. Rho kinase is thought to play a major role in cerebrovascular disease. The Rho kinase inhibitor, Y-27632, abolished augmented myogenic tone in middle cerebral arteries from S-P467L mice. CN-03, which modifies RhoA making it constitutively active, elevated myogenic tone to ≈60% in both strains, via a Y-27632–dependent mechanism. Large conductance Ca2+-activated K+ channels (BKCa) modulate myogenic tone. Inhibitors of BKCa caused greater constriction in middle cerebral arteries from nontransgenic littermates when compared with S-P467L. Expression of RhoA or Rho kinase-I/II protein was similar in cerebral arteries from S-P467L mice. Overall, the data suggest that peroxisome proliferator–activated receptor &ggr; in smooth muscle normally inhibits Rho kinase and promotes BKCa function, thus influencing myogenic tone in resistance arteries in brain. These findings have implications for mechanisms that underlie large- and small-vessel disease in brain, as well as regulation of cerebral blood flow.

Molecular mechanism of angiotensin II-induced insulin resistance in aortic vascular smooth muscle cells: Roles of Protein Tyrosine Phosphatase-1B

Insulin resistance is an underlying mechanism of type 2 diabetes and its vascular complications. Recent evidence suggests that crosstalk between angiotensin II (Ang II) and the insulin signaling in vascular smooth muscle cell (VSMC) may contribute to cellular insulin resistance. We hypothesized that Ang II inhibits the anti-mitogenic pathways while enhancing the mitogenic pathways stimulated by insulin via activation of Protein Tyrosine Phosphatase-1B (PTP-1B) in VSMC. We found that Ang II significantly inhibited insulin-induced phosphorylation of tyrosine 608 of IRS-1 and serine 473 of Akt, a downstream member of anti-mitogenic pathway of insulin. In contrast, Ang II increased the serine phosphorylation of IRS-1 which was not affected by the presence of insulin. Activation of p42/p44 MAPK (a mitogenic pathway) induced by insulin was further enhanced by Ang II. Transfection of VSMC with PTP-1B antisense oligonucleotide markedly reduced the effects of Ang II on insulin signaling. Furthermore, an increase in VSMC growth was attenuated by PTP-1B antisense only in the presence of both Ang II and insulin. Finally, we also showed that Ang II-induced activation of PTP-1B in VSMC was PKA/JAK2 dependent. We conclude that Ang II modulates both anti-mitogenic and mitogenic pathways of insulin via the activation of PTP-1B.

Interference with PPARγ in Vascular Smooth Muscle Causes Baroreflex Impairment and Autonomic Dysfunction

S-P467L mice expressing dominant negative peroxisome proliferator-activated receptor-&ggr; selectively in vascular smooth muscle exhibit impaired vasodilation, augmented vasoconstriction, hypertension, and tachycardia. We hypothesized that tachycardia in S-P467L mice is a result of baroreflex dysfunction. S-P467L mice displayed increased sympathetic traffic to the heart and decreased baroreflex gain and effectiveness. Carotid arteries exhibited inward remodeling but no changes in distensibility or stress/strain. Aortic depressor nerve activity in response to increased arterial pressure was blunted in S-P467L mice. However, the arterial pressure and heart rate responses to aortic depressor nerve stimulation were unaltered in S-P467L mice, suggesting that the central and efferent limbs of the baroreflex arc remain intact. There was no transgene expression in nodose ganglion and no change in expression of the acid-sensing ion channel-2 or -3 in nodose ganglion. There was a trend toward decreased expression of transient receptor potential vanilloid-1 receptor mRNA in nodose ganglion, but no difference in the immunochemical staining of transient receptor potential vanilloid-1 receptor in the termination area of the left aortic depressor nerve in S-P467L mice. Although there was no difference in the maximal calcium response to capsaicin in cultured nodose neurons from S-P467L mice, there was decreased desensitization of transient receptor potential vanilloid-1 receptor channels. In conclusion, S-P467L mice exhibit baroreflex dysfunction because of a defect in the afferent limb of the baroreflex arc caused by impaired vascular function, altered vascular structure, or compromised neurovascular coupling. These findings implicate vascular smooth muscle peroxisome proliferator activated receptor-&ggr; as a critical determinant of neurovascular signaling.

Role of PPARγ in Vascular Muscle in the Cerebral Circulation

Although peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) is thought to play a protective role in the vasculature, its cell-specific effect, particularly in resistance vessels, is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPAR&ggr; in vascular muscle would impair NO-dependent responses and augment vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPAR&ggr; (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPAR&ggr; in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses after interference with PPAR&ggr; involved Rho kinase with no apparent contribution by oxidative stress–related mechanisms. These findings support the concept that via effects on Rho kinase–dependent signaling, PPAR&ggr; in vascular muscle is a major determinant of vascular tone in resistance vessels and, in particular, NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow and the pathogenesis of large and small vessel disease in brain.Although peroxisome proliferator-activated receptor-gamma (PPARγ) is thought to play a protective role in the vasculature, its cell-specific impact, particularly in resistance vessels is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPARγ in vascular muscle would impair NO-dependent responses as well as augmenting vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPARγ (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPARγ in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses following interference with PPARγ involved Rho kinase with no apparent contribution by oxidative stress-related mechanisms. These findings support the concept that via effects on Rho kinase-dependent signaling, PPARγ in vascular muscle is a major determinant of vascular tone in resistance vessels, and in particular NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow as well as the pathogenesis of large and small vessel disease in brain.

Role of Peroxisome Proliferator–Activated Receptor-γ in Vascular Muscle in the Cerebral CirculationNovelty and Significance

Although peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) is thought to play a protective role in the vasculature, its cell-specific effect, particularly in resistance vessels, is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPAR&ggr; in vascular muscle would impair NO-dependent responses and augment vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPAR&ggr; (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPAR&ggr; in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses after interference with PPAR&ggr; involved Rho kinase with no apparent contribution by oxidative stress–related mechanisms. These findings support the concept that via effects on Rho kinase–dependent signaling, PPAR&ggr; in vascular muscle is a major determinant of vascular tone in resistance vessels and, in particular, NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow and the pathogenesis of large and small vessel disease in brain.Although peroxisome proliferator-activated receptor-gamma (PPARγ) is thought to play a protective role in the vasculature, its cell-specific impact, particularly in resistance vessels is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPARγ in vascular muscle would impair NO-dependent responses as well as augmenting vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPARγ (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPARγ in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses following interference with PPARγ involved Rho kinase with no apparent contribution by oxidative stress-related mechanisms. These findings support the concept that via effects on Rho kinase-dependent signaling, PPARγ in vascular muscle is a major determinant of vascular tone in resistance vessels, and in particular NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow as well as the pathogenesis of large and small vessel disease in brain.