GENETIC INTERFERENCE WITH PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ IN SMOOTH MUSCLE ENHANCES MYOGENIC TONE IN THE CEREBROVASCULATURE VIA A RHO KINASE-DEPENDENT MECHANISM

Abstract

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 γ (PPARγ) has emerged as a regulator of vascular tone. We hypothesized that interference with PPARγ in smooth muscle would augment myogenic responses in cerebral arteries. We studied transgenic mice expressing a dominant negative mutation in PPARγ selectively in smooth muscle (S-P467L) and nontransgenic littermates (non-Tg). Myogenic tone in middle cerebral arteries (MCA) from S-P467L was elevated 3-fold compared with non-Tg. Rho kinase is thought to play a major role in cerebrovascular disease. The Rho kinase inhibitor, Y-27632, abolished augmented myogenic tone in MCA 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 (BK_Ca) modulate myogenic tone. Inhibitors of BK_Ca caused greater constriction in MCA from non-Tg 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 PPARγ in smooth muscle normally inhibits Rho kinase and promotes BK_Ca 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.

Introduction

Consistent with the complexity of the organ, regulation of cerebral blood flow is highly developed involving multiple interacting cell types and molecular pathways^{1, 2}. One of the most important mechanisms contributing to the control of cerebral blood flow is autoregulation^{1, 3}. Maintenance of stable perfusion and coordinated delivery of nutrients to the brain parenchyma over a substantial range of perfusion pressures is achieved in large part by changes in myogenic tone¹. The myogenic response is an intrinsic property of vascular smooth muscle which translates changes in transmural pressure to changes in vessel diameter². Specifically, as intraluminal pressure increases or decreases, vessels constrict or dilate (respectively), thus contributing to the maintenance of blood flow to the brain parenchyma. Although cerebrovascular disease is often associated with changes in autoregulation with implications for brain perfusion, regulation of microvascular pressure, and permeability of the blood-brain barrier^{1, 4, 5}, our understanding of mechanisms that control these processes is limited.

Studies using pharmacological agonists [eg, thiazolidinediones (TZDs)] suggest that the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) exerts protective vascular effects in experimental models and patients with cardiovascular disease^6-8. In mice with a dominant negative mutation in PPARγ expressed selectively in smooth muscle (termed S-P467L), myogenic responses in mesenteric arteries are augmented in a protein kinase C (PKC)-dependent manner⁹. While autoregulation occurs in the mesenteric circulation, it is not as well developed as in the cerebral circulation^{1, 10}, where the calcium-sensitizing enzyme Rho kinase is thought to play a key role^{11, 12}. Furthermore, Rho kinase may be influenced by PPARγ¹³ and is thought to be of more importance than PKC in the regulation of myogenic tone during hypertension¹⁴.

Considering the importance of myogenic responses and recent evidence regarding the impact of PPARγ in vascular function, we tested the hypothesis that interference with PPARγ in smooth muscle would augment myogenic responses in cerebral arteries and examined mechanisms involved using several approaches. As Rho kinase is a key regulator of vascular tone in the cerebral circulation, we investigated the contribution of this signaling pathway in these responses. Our findings support the concept that Rho kinase has substantial effects on myogenic tone and that PPARγ in smooth muscle normally inhibits Rho kinase, influencing myogenic responses in resistance arteries in brain.

Materials and Methods

Please refer to Materials and Methods in the Online Supplement for the description of experimental procedures.

Results

Myogenic tone is selectively elevated in MCA from S-P467L mice

At 75 mmHg intraluminal pressure, myogenic tone was elevated approximately 3-fold in MCA from S-P467L mice compared with non-Tg controls (Figure 1A; P<0.05). By contrast, responses to 100 mmol/L KCl were similar indicating that overall contractility was similar between genotypes (Figure 1B). Active vessel diameter was reduced in MCA from S-P467L mice over a range of intraluminal pressures (Figure 1C; P<0.05). In contrast, passive vessel diameters (Ca²⁺-free conditions) were similar between genotypes (Figure 1D). As such, myogenic tone was elevated in MCA from S-P467L mice throughout this pressure range (Figure 1E; P<0.05), whereas tone remained relatively stable in MCA from non-Tg mice.

Figure 1.

(A) Myogenic tone of middle cerebral arteries (MCA) from non-transgenic (non-Tg) and S-P467L mice at 75 mmHg intraluminal pressure. (B) Response of MCA from non-Tg and S-P467L to 100 mmol/L KCl. (C) Active vessel diameters of MCA from non-Tg and S-P467L mice. (D) Passive vessel diameters of MCA from non-Tg and SP467L mice. (E) Steady-state myogenic tone in MCA from non-Tg and S-P467L. Constriction to KCl is expressed as % change in intraluminal diameter. Passive diameters were measured in Ca²⁺-free conditions. Myogenic tone was expressed relative to equivalent diameters in Ca²⁺-free conditions. (A) n=12 for non-Tg and n=15 for S-P467L, *P<0.05 vs. non-Tg (unpaired t test); (B) n=11 for non-Tg and S-P467L; (C) n=7 for non-Tg and S-P467L, *P<0.05 vs. non-Tg (two-way ANOVA); (D) n=7 for non-Tg and S-P467L. All data is expressed as mean±SE.

Evidence that elevated blood pressure in S-P467L mice does not contribute to augmented myogenic tone

S-P467L mice have a moderately elevated systolic blood pressure^{13, 15}. Thus, we considered the possibility that this modest elevation in blood pressure may contribute to the augmented myogenic tone observed in S-P467L mice (Figure 1A, 1E). To address this issue, we induced hypertension in non-Tg and SP467L mice using deoxycorticosterone acetate (DOCA) and 0.9% NaCl. Blood pressure was elevated in non-Tg mice (13±6 mmHg) and S-P467L (34±6 mmHg) following DOCA-salt treatment. The magnitude of the blood pressure increase in non-Tg was comparable to the moderately elevated blood pressure observed in S-P467L mice at baseline^{13, 15}. However, myogenic tone in DOCA-salt treated non-Tg was not altered compared to untreated controls (Figure 2). Furthermore, myogenic tone was similar between DOCA-salt treated and untreated S-P467L mice, suggesting that the elevation in myogenic tone occurs independently of a rise in arterial pressure (Figure 2).

Figure 2.

Myogenic tone of MCA from non-Tg and S-P467L mice at 75 mmHg intraluminal pressure. Deoxycorticosterone acetate (DOCA) and salt was used to induce hypertension prior to isolation of MCA for assessment of myogenic tone. Myogenic tone was expressed relative to equivalent diameters in Ca²⁺-free conditions. n=11 for untreated non-Tg, n=11 for untreated S-P467L, n=10 for DOCA treated non-Tg, n=11 for DOCA treated S-P467L, *P<0.05 vs. respective non-Tg (one-way ANOVA with a Tukey post-hoc test). All data is expressed as mean±SE.

No role for angiotensin type 1 or sphingosine-1-phosphate type 2 receptors in augmented myogenic tone

Following the generation of myogenic tone by MCA from S-P467L, we tested the effect of different inhibitors to evaluate potential molecular mechanisms that might underlie augmented myogenic tone. Neither the angiotensin type 1 receptor (AT1R) antagonist losartan nor the sphingosine-1-phosphate type 2 receptor (S1P2R) antagonist JTE-013 reduced myogenic tone in S-P467L mice (Figure S1).

Influence of reactive oxygen species (ROS) on myogenic tone in S-P467L mice

The superoxide dismutase (SOD) mimetic tempol reduced myogenic tone in MCA from S-P467L mice (Figure S2A; P<0.05), an effect that may be due to tempol-induced generation of hydrogen peroxide (H₂O₂), which dilates cerebral arteries¹⁶. Consistent with this possibility, treatment with EUK-134, a combined SOD and catalase mimetic, did not reduce myogenic tone generated by the MCA from S-P467L mice (Figure S2B).

PKC is not a mediator of the augmented myogenic tone in cerebral arteries

PKC can be an important regulator of vascular tone, and we obtained evidence previously that PKC mediates augmented myogenic tone in mesenteric arteries from S-P467L mice⁹. In contrast, treatment with the PKC inhibitor calphostin C (10 nmol/L), at a concentration that effectively reduced myogenic tone in mesenteric arteries from SP467L mice⁹, did not reduce myogenic tone in either non-Tg or S-P467L mice (Figure S3).

The RhoA/Rho kinase pathway plays a critical role in mediating augmented myogenic tone in S-P467L mice

We used two approaches to examine the impact of Rho kinase on myogenic tone. First, in MCA from S-P467L mice, treatment with a Rho kinase inhibitor (Y-27632) significantly reduced myogenic tone (Figure 3A: P<0.05). Treatment of MCA from non-Tg mice with Y-27632 reduced myogenic tone, but the overall level of tone was low and not statistically different (Figure 3A). Second, the influence of the RhoA/Rho kinase pathway on generation of myogenic tone was evaluated by examining effects of the direct RhoA activator CN-03. Treatment of MCA from either non-Tg of S-P467L with CN-03 (5 μg/ml) for 4 hours resulted in significantly augmented myogenic tone (~60% tone) at 75 mmHg compared with vehicle treated MCA (Figure 3B; P<0.05). Importantly, treatment with Y-27632 reduced tone in CN-03 treated MCA from non-Tg and S-P467L to ~10% of maximum diameter (Figure 3B; P<0.05).

Figure 3.

(A) Myogenic tone of MCA from non-Tg and S-P467L mice at 75 mmHg intraluminal pressure in the absence or presence of Y-27632. (B) Myogenic tone of MCA from non-Tg and S-P467L mice at 75 mmHg intraluminal pressure following treatment with either vehicle or CN-03. The effect of Y-27632 was tested in MCA treated with CN-03. Myogenic tone was expressed relative to equivalent diameters in Ca²⁺-free conditions. (A) *P<0.05 vs. control, n=7 (one-way ANOVA with a Tukey post-hoc test); (B) *P<0.05 vs. respective vehicle, #P<0.05 vs. respective CN-03, n=5 for all groups (one-way ANOVA with a Tukey post-hoc test). All data is expressed as mean±SE.

The influence of potassium channels is impaired in MCA from S-P467L mice

In arteries with established myogenic tone, treatment with an inhibitor of BK_Ca, iberiotoxin (a highly selective inhibitor of this channel), resulted in a concentration-dependent increase in vascular tone in both genotypes. However, the magnitude of tone generated in the MCA from S-P467L was significantly less compared with non-Tg (P<0.05; Figure 4). Consistently, a second BK_Ca inhibitor TEA induced less constriction in the MCA from S-P467L compared with non-Tg (Figure S4; P<0.05).

Figure 4.

Constriction of MCA from non-Tg and S-P467L mice to iberiotoxin. *P<0.05 vs. non-Tg at the respective concentration, (one-way ANOVA with a Tukey post-hoc test); n=5. All data is expressed as mean±SE.

mRNA expression of components of the Rho kinase pathway are not altered in cerebral arteries from S-P467L mice

We found no significant change in mRNA levels of RhoA, Rho kinase isoforms 1 and 2 (ROCK-I and ROCK-II), regulator of G-protein signaling (RGS) 2 and 5 and the BK_Ca subunits KCNMA1 and KCNMB1 in cerebral arteries from S-P467L mice compared with non-Tg (Table S1).

Protein expression in cerebral arteries

Total PPARγ protein expression was significantly elevated in cerebral arteries from S-P467L mice compared with non-Tg (Figure S5). This finding is consistent with our previous measurements in this model⁹, which overexpresses dominant-negative PPARγ in vascular muscle. We next performed protein expression analysis on RhoA and ROCK-I and ROCK-II. There was a trend for a small increase in RhoA protein expression, however this did not reach statistical significance (Figure 5A; P=0.12). Protein expression of ROCK-I and ROCK-II was similar between groups (Figure 5B-C).

Figure 5.

Protein expression of RhoA (A), Rho kinase I (ROCK-I; B) and ROCK-II (C) in cerebral arteries from non-Tg and S-P467L mice. (A) n=6, (B) n=5 and (C) n=9. Protein expression was normalized to expression of β-actin. All data is expressed as mean±SE.

Because post-translational modification of myosin light chain phosphatase (MLCP) can alter protein activity without a change in protein expression, we measured the phosphorylation state of the regulatory subunit of MLCP, MYPT1. We found no consistent change in the basal phosphorylation state of MYPT1 at either Thr⁶⁹⁶ or Thr⁸⁵³ (Figure S6).

Discussion

In this study, we used a genetic approach and cell-specific manipulation of PPARγ to examine mechanisms that influence myogenic responses in cerebral blood vessels. There are several major new findings. The data support the concept that PPARγ in vascular muscle plays a key role in regulating myogenic responses in the cerebral circulation, an organ which normally autoregulates very effectively. Specifically, we report that the MCA from S-P467L mice generate significantly more myogenic tone compared with non-Tg and this effect is independent of modest elevations in arterial pressure. Furthermore, our findings suggest that the augmented myogenic tone is dependent on the RhoA/Rho kinase signaling pathway, but is not mediated by changes in RhoA or Rho kinase protein expression. We also provide evidence that impaired BK_Ca function may contribute to the augmented myogenic response in S-P467L mice. Overall, these findings highlight a critical role for PPARγ in vascular muscle modulating the activity of the RhoA/Rho kinase pathway and thus, influencing myogenic responses.

Myogenic tone and myogenic responses of cerebral arteries and arterioles play a key role in maintaining proper cerebral perfusion while also protecting the downstream microcirculation and blood-brain barrier from potentially detrimental elevations in local intravascular pressure². Cerebral arteries are an important site of vascular resistance in brain⁵. In the present study, genetic interference with the normal activity of PPARγ in cerebrovascular muscle resulted in a significant increase in myogenic tone. This elevation in myogenic tone was observed over a range of physiologically relevant intraluminal pressures. By contrast, responses to a high concentration of KCl were not affected by PPARγ interference. Thus, in the absence of any other intervention, PPARγ in vascular muscle normally protects against potentially excessive increases in myogenic tone. Previous work using systemic TZD treatment suggested that PPARγ can influence myogenic tone in cerebral arteries in a model of hypertension¹⁷. The limitations of using TZDs include the fact that they lack cell specificity and do not provide insight into the impact of PPARγ when activated by endogenous ligands. Thus, the current findings extend this concept demonstrating that PPARγ in vascular muscle has a prominent effect on myogenic tone under normal conditions (in the absence of TZD treatment).

We found no difference in inner diameter of the MCA in Ca²⁺ free conditions suggesting that no remodeling had occurred in these arteries from S-P467L mice. This observation contrasts with the inward remodeling seen in smaller downstream pial arterioles from the same mice,¹³ as well as the same arterioles in mice that express the P465L mutation in all cells¹⁸. In contrast, but in agreement with our data in the MCA, there was no change in inner diameter of small mesenteric arteries (a vessel of similar size to the MCA used in the present study) from S-P467L mice⁹. Overall, the results suggest that PPARγ differentially affects vessel structure depending on location within the vascular tree.

It has been shown that patients with dominant-negative mutations in PPARγ present with early onset hypertension, insulin resistance and diabetes mellitus^{19, 20}. Consistent with the smooth muscle specific manipulation used, S-P467L mice have a modest elevation in blood pressure but no change in body weight, adipose tissue depots or plasma glucose, insulin, or leptin^{13, 15}. To determine if this elevated blood pressure might account for augmented myogenic tone, we also studied mice treated with DOCA-salt. This model of hypertension exhibits a modest elevation in blood pressure of ~10-15 mmHg in wild type mice, similar to the elevation in blood pressure in S-P467L mice at baseline¹³. Despite the elevation in arterial pressure, non-Tg mice treated with DOCA-salt had comparable myogenic tone to that seen in untreated non-Tg. Furthermore, DOCA-salt treatment did not further increase myogenic tone in S-P467L mice. We treated non-Tg and S-P467L mice with DOCA-salt for 21 days. While we were able to match levels or arterial pressure, the time course of hypertension in SP467L mice is currently unknown. Thus, while it is possible that the duration of hypertension was different in the two models, we are not aware of data in the literature demonstrating that myogenic tone continues to change over time if blood pressure is stable. Despite this potential limitation, our findings suggest that the large difference in myogenic tone in S-P467L mice is likely due predominantly to the effect of dominant negative PPARγ in smooth muscle, and not a secondary response to the moderate elevation in blood pressure caused by this mutation.

It was recently reported that angiotensin II-induced hypertension increases myogenic tone²¹. The difference between that previous work and the present study may be explained by approaches used to elevate blood pressure (angiotensin II vs DOCA-salt) as well as the magnitude of pressure elevation (~50 mmHg vs ~15 mmHg)²¹. We suggest that the modest elevation in blood pressure produced in the current study is insufficient to significantly augment myogenic tone in the MCA at physiological pressures. However, we cannot exclude the possibility that more extreme portions of the myogenic response curve are changed.

Despite being studied for over 100 years, many aspects of the myogenic response are still not well defined and often debated including the identity of the mechanosensor(s) that couples changes in intraluminal pressure to vascular tone (reviewed in^{2, 22}). Although both AT1 and S1P2 receptors have been implicated in some models^23-25, we found no role for these receptors in mediating augmented myogenic tone in cerebral arteries from S-P467L mice. This finding related to AT1 receptors is consistent with other studies in which antagonists of AT1 receptors did not affect diameter of cerebral arterioles with myogenic tone^{26, 27}. The possibility remains that these receptors influence myogenic tone under other conditions.

Rho kinase is thought to be an important contributor to vascular disease progression and as such, a potential therapeutic target²⁸. We found previously that vascular dysfunction in aorta following genetic interference with PPARγ in smooth muscle is due to decreased RhoA turnover and thus increased activation of Rho kinase¹⁵. Whether this same mechanism is functionally important in resistance vessels was unclear however as increased myogenic tone in mesenteric arteries from S-P467L mice was mediated by PKC and not Rho kinase⁹. In contrast, we found no role for PKC in mediating myogenic tone in cerebral arteries from S-P467L mice. These findings suggest that despite a similar phenotype (increased myogenic tone in both mesenteric and cerebral arteries), the underlying mechanism that produced these effects in SP467L mice is fundamentally different. The present study suggests that PPARγ in vascular muscle has a substantial influence on myogenic tone in cerebral arteries (resistance vessels in brain) and provides evidence this effect occurs via modulation of the RhoA/Rho kinase pathway. Two lines of evidence supported the concept that Rho kinase influences myogenic tone. First, a selective Rho kinase inhibitor Y-27632 reversed augmented myogenic tone in the MCA from S-P467L mice. Secondly, a direct activator of RhoA (CN-03) caused significant augmentation in myogenic tone in both genotypes that was Rho kinase dependent. The active site of CN-03 is designed to mimic the catalytic domain of the cytotoxic necrotizing factor-1 (CNF-1) 1 from E. coli. In a similar manner to CNF-1 toxin, this cell permeable compound deamidates glutamine at position 63 on RhoA, converting it to a glutamate^{29, 30}. This change disrupts both the GAP-stimulated and intrinsic GTPase activity of RhoA making it constitutively active^{29, 30} resulting in increased activation of Rho kinase and greater myogenic tone.

Rho kinase plays a crucial role in regulating contractility of vascular muscle via effects on calcium sensitivity. This effect is achieved by phosphorylating the regulatory subunit of MLCP (MYPT1), which results in inhibition of MLCP activity and maintenance of force due to reduced de-phosphorylation of myosin³¹. While we were able to detect phosphorylated MYPT1, we could not consistently detect significant changes in phosphorylated MYPT1 at either Thr⁶⁹⁶ or Thr⁸⁵³. One possible explanation of this is that vessels used for western blotting were not pressurized, so the results may reflect ‘basal’ MYPT1 phosphorylation. Thus, while we were unable to detect changes in a Rho kinase target in mouse cerebral arteries, other lines of evidence suggest that augmented myogenic tone in S-P467L mice is Rho kinase dependent.

We next attempted to elucidate a possible mechanism by which the Rho kinase pathway was being activated. Because ROS may activate RhoA/Rho kinase in vascular tissue^{32, 33}, we used a superoxide dismutase mimetic (tempol) and a combined superoxide-H₂O₂ scavenger (EUK-134). Although tempol reduced myogenic tone in SP467L mice by ~30%, EUK-134 had no effect on myogenic tone in arteries from SP467L mice. Thus, the tempol-induced reduction in tone may result from vasodilation in response to H₂O₂ generated by tempol and not direct inhibition of myogenic tone. While ROS are believed to be important regulators of cerebrovascular tone, our findings suggest that ROS are unlikely to play a major role in augmented myogenic tone in SP467L mice.

Potassium channels in vascular muscle have significant effects on myogenic tone^{34, 35}. For example, activation of BK_Ca opposes increases in vascular tone³⁶. As intracellular levels of Ca²⁺ increase, BK_Ca are activated, resulting in hyperpolarization of the cell, closure of voltage-dependent Ca²⁺ channels and vasodilation³⁴. Inhibition of BK_Ca increases vascular tone by disruption of this modulatory response. Consistent with this concept and our findings in mesenteric arteries⁹, we found that inhibition of BK_Ca with either iberiotoxin or TEA resulted in a greater constriction in arteries from non-Tg compared with S-P467L mice. Messenger RNA expression of the BK_Ca subunits KCNMA1 and KCNMB1 were not changed in cerebral arteries from S-P467L mice. Thus, interference with PPARγ in vascular muscle alters the influence of BK_Ca on vascular tone, an important feedback mechanism that modulates myogenic responses. We considered the possibility that disruption of BK_Ca function increases RhoA/Rho kinase signaling. It has previously been shown that membrane depolarization using 60 mmol/L K⁺ causes contraction of vascular muscle via Ca²⁺-dependent activation of RhoA/Rho kinase^{37, 38}. However, the degree of membrane depolarization produced by inhibition of BK_Ca function is likely much less than that produced by high concentrations of potassium. Thus, it is unclear if BK_Ca dysfunction in S-P467L mice was sufficient to augment RhoA/Rho kinase activity and thus myogenic tone.

We attempted to gain additional insight into the molecular basis for changes in myogenic tone in S-P467L mice. Real time PCR revealed no differences in mRNA expression of components of the RhoA/Rho kinase signaling pathway (RhoA and Rho kinase I and II) in cerebral arteries. RGS5 has been identified as a potential regulator of vascular function^{9, 39}. Although expression of RGS5 is reduced in mesenteric arteries from S-P467L mice⁹, RGS5 mRNA levels were not affected in cerebral arteries. Expression of RhoA and Rho kinase isoforms at the protein level were also unchanged in cerebral arteries from S-P467L mice. Our finding that there was only a small increase in RhoA protein level is in contrast with our previous finding in aorta, where we obtained evidence that vascular dysfunction in S-P467L mice was due to decreased RhoA turnover¹⁵. Our findings in the current study using CN-03 support the concept of no change in RhoA expression in cerebral arteries, as the maximum tone generated was similar between genotypes, suggesting the amount of RhoA available to be modified by the toxin was similar. Both the present and previous studies suggest that PPARγ regulates key regulatory pathways in vascular muscle, however the underlying molecular mechanism by which it exerts these effects differ between vascular beds. Overall, the findings of the present study and others¹⁵ suggest interference with PPARγ affects activity of Rho kinase without major changes in protein expression, a finding consistent with other models^{15, 40}.

In conclusion, we have obtained genetic and pharmacological evidence that PPARγ in vascular muscle has a significant influence on myogenic tone in brain via suppression of RhoA/Rho kinase-dependent signaling as well as an influence on function of BK_Ca. Our findings add to the growing evidence that PPARγ in vascular cells influence key signaling pathways and thus are an important contributor to vascular homeostasis.

Perspective

The brain autoregulates very effectively via mechanisms that are impacted by disease. Although myogenic responses in cerebral vessels have been widely studied (for example^{17, 41}), the use of cell-specific manipulation to define the impact of specific molecules or pathways has been relatively rare. The current data based on selective genetic interference with PPARγ in smooth muscle highlights the importance of this molecule as a determinant of myogenic tone in the cerebrovasculature. Our findings suggest that PPARγ in vascular muscle normally inhibits myogenic tone via mechanisms that involve suppression of Rho kinase signaling. Such a mechanism may be protective by limiting excessive increases in myogenic tone that could contribute to reductions in cerebral blood flow (hypoperfusion), implicated in conditions including small vessel disease and cognitive decline^{4, 42}. Inhibition of Rho kinase by PPARγ is also potentially significant because activity of Rho kinase is positively associated with cardiovascular events including stroke⁴³.

Novelty and Significance.

1. What Is New?

• A prominent influence of PPARγ on myogenic tone in cerebral arteries was seen under normal conditions, in the absence of treatment with exogenous activators of PPARγ.

• These findings support the concept that Rho kinase has substantial effects on myogenic tone and that PPARγ in vascular muscle normally inhibits Rho kinase-dependent signaling, thus influencing myogenic tone in resistance arteries in brain.

2. What Is Relevant?

• While autoregulation is considered to be one of the most important processes regulating cerebral blood flow, mechanisms that underlie this response at the cell-specific level are still poorly defined.

• Cerebrovascular disease is often associated with changes in autoregulation, but our understanding of mechanisms that control these processes is limited.

3. Summary

This study provides genetic and pharmacological evidence that PPARγ in vascular muscle has a significant influence on myogenic tone in brain via suppression of RhoA/Rho kinase-dependent signaling. Interference with PPARγ signaling in smooth muscle also alters the influence of BK_Ca on cerebrovascular function. These findings add to the growing evidence that PPARγ in vascular cells influences key signaling pathways and thus are important contributors to vascular homeostasis.