T. Michael De Silva

Cerebral Vascular Disease and Neurovascular Injury in Ischemic Stroke

The consequences of cerebrovascular disease are among the leading health issues worldwide. Large and small cerebral vessel disease can trigger stroke and contribute to the vascular component of other forms of neurological dysfunction and degeneration. Both forms of vascular disease are driven by diverse risk factors, with hypertension as the leading contributor. Despite the importance of neurovascular disease and subsequent injury after ischemic events, fundamental knowledge in these areas lag behind our current understanding of neuroprotection and vascular biology in general. The goal of this review is to address select key structural and functional changes in the vasculature that promote hypoperfusion and ischemia, while also affecting the extent of injury and effectiveness of therapy. In addition, as damage to the blood–brain barrier is one of the major consequences of ischemia, we discuss cellular and molecular mechanisms underlying ischemia-induced changes in blood–brain barrier integrity and function, including alterations in endothelial cells and the contribution of pericytes, immune cells, and matrix metalloproteinases. Identification of cell types, pathways, and molecules that control vascular changes before and after ischemia may result in novel approaches to slow the progression of cerebrovascular disease and lessen both the frequency and impact of ischemic events.

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.

Heterogeneous Impact of ROCK2 on Carotid and Cerebrovascular Function.

Rho kinase (ROCK) has been implicated in physiological and pathophysiological processes, including regulation of vascular function. ROCK signaling is thought to be a critical contributor to cardiovascular disease, including hypertension and effects of angiotensin II (Ang II). Two isoforms of ROCK (1 and 2) have been identified and are expressed in vascular cells. In this study, we examined the importance of ROCK2 in relation to vessel function using several models and a novel inhibitor of ROCK2. First, incubation of carotid arteries with the direct RhoA activator CN-03 or Ang II impaired endothelium-dependent relaxation by ≈40% to 50% (P<0.05) without altering endothelium-independent relaxation. Both CN-03- and Ang II–induced endothelial dysfunction was prevented by Y-27632 (an inhibitor of both ROCK isoforms) or the selective ROCK2 inhibitor SLX-2119. In contrast, SLX-2119 had little effect on contraction of carotid arteries to receptor-mediated agonists (serotonin, phenylephrine, vasopressin, or U46619). Second, in basilar arteries, SLX-2119 inhibited constriction to Ang II by ≈90% without significantly affecting responses to serotonin or KCl. Third, in isolated pressurized brain parenchymal arterioles, SLX-2119 inhibited myogenic tone in a concentration-dependent manner (eg, 1 &mgr;mol/L SLX-2119 dilated by 79±4%). Finally, SLX-2119 dilated small pial arterioles in vivo, an effect that was augmented by inhibition of nitric oxide synthase. These findings suggest that ROCK2 has major, but heterogeneous, effects on function of endothelium and vascular muscle. The data support the concept that aberrant ROCK2 signaling may be a key contributor to select aspects of large and small vessel disease, including Ang II–induced endothelial dysfunction.

Protective Role for Tissue Inhibitor of Metalloproteinase-4, a Novel Peroxisome Proliferator–Activated Receptor-γ Target Gene, in Smooth Muscle in Deoxycorticosterone Acetate–Salt Hypertension

Loss of peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) function causes hypertension, whereas its activation lowers blood pressure. Evidence suggests that these effects may be attributable to PPAR&ggr; activity in the vasculature. However, the specific transcriptional targets of PPAR&ggr; in vessels remain largely unidentified. In this study, we examined the role of smooth muscle PPAR&ggr; during salt-sensitive hypertension and investigated its transcriptional targets and functional effect. Transgenic mice expressing dominant-negative PPAR&ggr; (S-P467L) in smooth muscle cells were more prone to deoxycorticosterone acetate–salt–induced hypertension and mesenteric arterial dysfunction compared with nontransgenic controls. Despite similar morphometry at baseline, vascular remodeling in conduit and small arteries was enhanced in S-P467L after deoxycorticosterone acetate–salt treatment. Gene expression profiling in aorta and mesenteric arteries revealed significantly decreased expression of tissue inhibitor of metalloproteinase-4 (TIMP-4) in S-P467L. Expression of TIMP-4 was increased by deoxycorticosterone acetate–salt treatment, but this increase was ablated in S-P467L. Interference with PPAR&ggr; activity either by treatment with a PPAR&ggr; inhibitor, GW9662, or by expressing P467L PPAR&ggr; markedly suppressed TIMP-4 in primary smooth muscle cells. PPAR&ggr; binds to a PPAR response element (PPRE) in chromatin close to the TIMP-4 gene in smooth muscle cells, suggesting that TIMP-4 is a novel target of PPAR&ggr;. The interference with PPAR&ggr; and decrease in TIMP-4 were accompanied by an increase in total matrix metalloproteinase activity. PPAR&ggr;-mediated loss of TIMP-4 increased, whereas overexpression of TIMP-4 decreased smooth muscle cell migration in a scratch assay. Our findings highlight a protective mechanism induced by PPAR&ggr; in deoxycorticosterone acetate–salt treatment, establishing a novel mechanistic link between PPAR&ggr; and TIMP-4.

Selective inhibition of brain endothelial Rho-kinase-2 provides optimal protection of an in vitro blood-brain barrier from tissue-type plasminogen activator and plasmin

Rho-kinase (ROCK) inhibition, broadly utilised in cardiovascular disease, may protect the blood-brain barrier (BBB) during thrombolysis from rt-PA-induced damage. While the use of nonselective ROCK inhibitors like fasudil together with rt-PA may be hindered by possible hypotensive side-effects and inadequate capacity to block detrimental rt-PA activity in brain endothelial cells (BECs), selective ROCK-2 inhibition may overcome these limitations. Here, we examined ROCK-2 expression in major brain cells and compared the ability of fasudil and KD025, a selective ROCK-2 inhibitor, to attenuate rt-PA-induced BBB impairment in an in vitro human model. ROCK-2 was highly expressed relative to ROCK-1 in all human and mouse brain cell types and particularly enriched in rodent brain endothelial cells and astrocytes compared to neurons. KD025 was more potent than fasudil in attenuation of rt-PA- and plasminogen-induced BBB permeation under normoxia, but especially under stroke-like conditions. Importantly, only KD025, but not fasudil, was able to block rt-PA-dependent permeability increases, morphology changes and tight junction degradation in isolated BECs. Selective ROCK-2 inhibition further diminished rt-PA-triggered myosin phosphorylation, shape alterations and matrix metalloprotease activation in astrocytes. These findings highlight ROCK-2 as the key isoform driving BBB impairment and brain endothelial damage by rt-PA and the potential of KD025 to optimally protect the BBB during thrombolysis.

Genetic Interference With Endothelial PPAR-γ (Peroxisome Proliferator–Activated Receptor-γ) Augments Effects of Angiotensin II While Impairing Responses to Angiotensin 1–7Novelty and Significance

Pharmacological activation of PPAR-&ggr; (peroxisome proliferator–activated receptor-&ggr;) protects the vasculature. Much less is known on the cell-specific impact of PPAR-&ggr; when driven by endogenous ligands. Recently, we found that endothelial PPAR-&ggr; protects against angiotensin II–induced endothelial dysfunction. Here, we explored that concept further examining whether effects were sex dependent along with underlying mechanisms. We studied mice expressing a human dominant–negative mutation in PPAR-&ggr; driven by the endothelial-specific vascular cadherin promoter (E-V290M), using nontransgenic littermates as controls. Acetylcholine (an endothelium-dependent agonist) produced similar relaxation of carotid arteries from nontransgenic and E-V290M mice. Incubation of isolated arteries with angiotensin II (1 nmol/L) overnight had no effect in nontransgenic, but reduced responses to acetylcholine by about 50% in male and female E-V290M mice (P<0.05). Endothelial function in E-V290M mice was restored to normal by inhibitors of superoxide (tempol), NADPH oxidase (VAS-2870), Rho kinase (Y-27632), ROCK2 (SLX-2119), NF-&kgr;B (nuclear factor-kappa B essential modulator–binding domain peptide), or interleukin-6 (neutralizing antibody). In addition, we hypothesized that PPAR-&ggr; may influence the angiotensin 1–7 arm of the renin–angiotensin system. In the basilar artery, dilation to angiotensin 1–7 was selectively reduced in E-V290M mice by >50% (P<0.05), an effect reversed by Y-27632. Thus, effects of angiotensin II are augmented by interference with endothelial PPAR-&ggr; through sex-independent mechanisms, involving oxidant–inflammatory signaling and ROCK2 (Rho kinase). The study also provides the first evidence that endothelial PPAR-&ggr; interacts with angiotensin 1–7 responses. These critical roles for endothelial PPAR-&ggr; have implications for pathophysiology and therapeutic approaches for vascular disease.

Changes in Cerebral Arteries and Parenchymal Arterioles With Aging: Role of Rho Kinase 2 and Impact of Genetic Background

Vascular aging fundamentally contributes to large and small vessel disease. Despite the importance of such changes for brain function, mechanisms that mediate such changes are poorly defined. We explored mechanisms that underlie changes with age, testing the hypothesis that ROCK (Rho kinase) plays an important role. In C57BL/6 mice, baseline diameters of isolated pressurized parenchymal arterioles were similar in adult (4–5 month) and old mice (22±1 month; ≈15±1 µm). Endothelium-dependent dilation was impaired in old mice compared with adults in a pathway-specific manner. Vasodilation to NS-309 (which activates small- and intermediate-conductance Ca2+ activated K+ channels in endothelial cells) was intact while endothelial nitric oxide synthase–mediated vasodilation was reduced by ≥60%, depending on the concentration (P<0.05). A similar reduction was present in basilar arteries. Inhibiting both ROCK isoforms with Y-27632 restored the majority of endothelial function in old mice. Because genetic background is a determinant of vascular disease, we performed similar studies using FVB/N mice. Endothelial dysfunction was seen with aging in both FVB/N and C57BL/6 mice although the magnitude was increased ≈2-fold in the latter strain (P<0.05). In both strains of mice, age-induced endothelial dysfunction was reversed by inhibition of ROCK2 with SLX-2119. Thus, aging impairs endothelial function in both cerebral arteries and parenchymal arterioles, predominantly via effects on endothelial nitric oxide synthase–dependent regulation of vascular tone. The magnitude of these changes was influenced by genetic background and mediated by ROCK2.

Endothelial PPARγ (Peroxisome Proliferator–Activated Receptor-γ) Is Essential for Preventing Endothelial Dysfunction With AgingNovelty and Significance

Little is known about mechanisms that control vascular aging, particularly at the cell-specific level. PPAR&ggr; (peroxisome proliferator–activated receptor-&ggr;) exerts protective effects in the vasculature when activated pharmacologically. To gain insight into the cell-specific impact of PPAR&ggr;, we examined the hypothesis that genetic interference with endothelial PPAR&ggr; would augment age-induced vascular dysfunction. We studied carotid arteries from adult (11.6±0.3 months) and old (24.7±0.6 months) mice with endothelial-specific expression of a human dominant negative mutation in PPAR&ggr; driven by the vascular cadherin promoter (E-V290M), along with age-matched, nontransgenic littermates. Acetylcholine (an endothelium-dependent agonist) produced similar relaxation in arteries from adult nontransgenic and E-V290M mice and old nontransgenic mice. In contrast, responses to acetylcholine were reduced by >50% in old male and female E-V290M mice (P<0.01). Endothelial function in old E-V290M mice was not altered by an inhibitor of COX (cyclooxygenase) but was restored to normal by a superoxide scavenger, an inhibitor of NADPH oxidase, or inhibition of ROCK (Rho kinase). Relaxation of arteries to nitroprusside, which acts directly on vascular muscle, was similar in all groups. Vascular expression of IL (interleukin)-6, Nox-2, and CDKN2A (a marker of senescence) was significantly increased in old E-V290M mice compared with controls (P<0.05). These findings provide the first evidence that age-related vascular dysfunction, inflammation, and senescence is accelerated after interference with endothelial PPAR&ggr; via mechanisms involving oxidative stress and ROCK. The finding of an essential protective role for endothelial PPAR&ggr; has implications for vascular disease and therapy for vascular aging.

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.