Christopher J. Pelham

Does Peroxisome Proliferator-activated Receptor-γ (PPARγ) Protect from Hypertension Directly through Effects in the Vasculature?

Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor of the nuclear hormone receptor superfamily. Increasing evidence suggests that PPARγ is involved in the regulation of vascular function and blood pressure in addition to its well recognized role in metabolism. Thiazolidinediones, PPARγ agonists, lower blood pressure and have protective vascular effects through largely unknown mechanisms. In contrast, loss-of-function dominant-negative mutations in human PPARγ cause insulin resistance and severe early onset hypertension. Recent studies using genetically manipulated mouse models have begun to specifically address the importance of PPARγ in the vasculature. In this minireview, evidence for a protective role of PPARγ in the endothelium and vascular smooth muscle, derived largely from studies of genetically manipulated mice, will be discussed.

PPARγ Regulates Resistance Vessel Tone Through a Mechanism Involving RGS5-Mediated Control of Protein Kinase C and BKCa Channel Activity

Rationale: Activation of peroxisome proliferator−activated receptor-&ggr; (PPAR&ggr;) by thiazolidinediones lowers blood pressure, whereas PPAR&ggr; mutations cause hypertension. Previous studies suggest these effects may be mediated through the vasculature, but the underlying mechanisms remain unclear. Objective: To identify PPAR&ggr; mechanisms and transcriptional targets in vascular smooth muscle and their role in regulating resistance artery tone. Methods and Results: We studied mesenteric artery (MA) from transgenic mice expressing dominant-negative (DN) mutant PPAR&ggr; driven by a smooth muscle cell−specific promoter. MA from transgenic mice exhibited a robust increase in myogenic tone. Patch clamp analysis revealed a reduced large conductance Ca2+-activated K+ (BKCa) current in freshly dissociated smooth muscle cell from transgenic MA. Inhibition of protein kinase C corrected both enhanced myogenic constriction and impaired the large conductance Ca2+-activated K+ channel function. Gene expression profiling revealed a marked loss of the regulator of G protein signaling 5 (RGS5) mRNA in transgenic MA, which was accompanied by a substantial increase in angiotensin II–induced constriction in MA. Small interfering RNA targeting RGS5 caused augmented myogenic tone in intact mesenteric arteries and increased activation of protein kinase C in smooth muscle cell cultures. PPAR&ggr; and PPAR&dgr; each bind to a PPAR response element close to the RGS5 promoter. RGS5 expression in nontransgenic MA was induced after activation of either PPAR&ggr; or PPAR&dgr;, an effect that was markedly blunted by DN PPAR&ggr;. Conclusions: We conclude that RGS5 in smooth muscle is a PPAR&ggr; and PPAR&dgr; target, which when activated blunts angiotensin II–mediated activation of protein kinase C, and preserves the large conductance Ca2+-activated K+ channel activity, thus providing tight control of myogenic tone in the microcirculation.

Preservation of Intracellular Renin Expression Is Insufficient to Compensate for Genetic Loss of Secreted Renin

The primary product of the renin gene is preprorenin. A signal peptide sorts renin to the secretory pathway in juxtaglomerular cells where it is released into the circulation to initiate the renin-angiotensin system cascade. In the brain, transcription of renin occurs from an alternative promoter encoding an mRNA starting with a new first exon (exon 1b). Exon 1b initiating transcripts skip over the classical first exon (exon 1a) containing the initiation codon for preprorenin. Exon 1b transcripts are predicted to use a highly conserved initiation codon within exon 2, producing renin, which should remain intracellular, because it lacks the signal peptide. To evaluate the roles of secreted and intracellular renin, we took advantage of the organization of the renin locus to generate a secreted renin (sRen)-specific knockout, which preserves intracellular renin expression. Expression of sRen mRNA was ablated in the brain and kidney, whereas intracellular renin mRNA expression was preserved in fetal and adult brains. We noted a developmental shift from the expression of sRen mRNA in the fetal brain to intracellular renin mRNA in the adult brain. Homozygous sRen knockout mice exhibited very poor survival at weaning. The survivors exhibited renal lesions, low hematocrit, an inability to generate a concentrated urine, decreased arterial pressure, and impaired aortic contraction. These results suggest that preservation of intracellular renin expression in the brain is not sufficient to compensate for a loss of sRen, and sRen plays a pivotal role in renal development and function, survival, and the regulation of arterial pressure.

Pregnant mice lacking indoleamine 2,3-dioxygenase exhibit preeclampsia phenotypes

Preeclampsia is a cardiovascular disorder of late pregnancy that is, commonly characterized by hypertension, renal structural damage and dysfunction, and fetal growth restriction. Prevailing etiologic models of this disorder include T‐cell dysfunction as an initiating cause of preeclampsia. Indoleamine 2,3‐dioxygenase (IDO), an enzyme that mediates the conversion of tryptophan to kynurenine, has been linked to preeclampsia in humans, and is known to regulate T‐cell activity and an endothelial‐derived relaxing factor. To test the hypothesis that IDO is causally involved in the pathogenesis of preeclampsia, mice deficient for IDO (IDO‐KO) were generated on a C57BL/6 background. IDO‐KO and wild‐type C57BL/6 mice were bred, and preeclampsia phenotypes were evaluated during pregnancy. Pregnant IDO‐KO mice exhibited pathognomonic renal glomerular endotheliosis, proteinuria, pregnancy‐specific endothelial dysfunction, intrauterine growth restriction, and mildly elevated blood pressure compared to wild‐type mice. Together these findings highlight an important role for IDO in the generation of phenotypes typical of preeclampsia. Loss of IDO function may represent a risk factor for the development of preeclampsia. By extension, increased IDO activity, reductions in IDO reactants, or increases in IDO products may represent novel therapeutic approaches for this disorder.

Dominant negative PPARγ promotes atherosclerosis, vascular dysfunction, and hypertension through distinct effects in endothelium and vascular muscle

Agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) have potent insulin-sensitizing effects and inhibit atherosclerosis progression in patients with Type II diabetes. Conversely, missense mutations in the ligand-binding domain of PPARγ that render the transcription factor dominant negative (DN) cause early-onset hypertension and Type II diabetes. We tested the hypothesis that DN PPARγ-mediated interference of endogenous wild-type PPARγ in the endothelium and vascular smooth muscle exacerbates atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-specific expression of DN PPARγ on the ApoE(-/-) background unmasked significant impairment of endothelium-dependent relaxation in aortic rings, increased systolic blood pressure, altered expression of atherogenic markers (e.g., Cd36, Mcp1, Catalase), and enhanced diet-induced atherosclerotic lesion formation in aorta. Smooth muscle-specific expression of DN PPARγ, which induces aortic dysfunction and increased systolic blood pressure at baseline, also resulted in enhanced diet-induced atherosclerotic lesion formation in aorta on the ApoE(-/-) background that was associated with altered expression of a shared, yet distinct, set of atherogenic markers (e.g., Cd36, Mcp1, Osteopontin, Vcam1). In particular, induction of Osteopontin expression by smooth muscle-specific DN PPARγ correlated with increased plaque calcification. These data demonstrate that inhibition of PPARγ function specifically in the vascular endothelium or smooth muscle may contribute to cardiovascular disease.

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.

MAP/ERK kinase kinase 1 (Mekk1) mediates transcriptional repression by interacting with polycystic kidney disease-1 (PKD1) promoter-bound p53 tumor suppressor protein

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

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.

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.