Milton Hamblin

Loss of Perivascular Adipose Tissue on Peroxisome Proliferator–Activated Receptor-γ Deletion in Smooth Muscle Cells Impairs Intravascular Thermoregulation and Enhances Atherosclerosis

Background— Perivascular adipose tissue (PVAT) surrounds most vessels and shares common features with brown adipose tissue (BAT). Although adaptive thermogenesis in BAT increases energy expenditure and is beneficial for metabolic diseases, little is known about the role of PVAT in vascular diseases such as atherosclerosis. We hypothesize that the thermogenic function of PVAT regulates intravascular temperature and reduces atherosclerosis. Methods and Results— PVAT shares similar structural and proteomics with BAT. We demonstrated that PVAT has thermogenic properties similar to BAT in response to cold stimuli in vivo. Proteomics analysis of the PVAT from mice housed in a cold environment identified differential expression in proteins highly related to cellular metabolic processes. In a mouse model deficient in peroxisome proliferator–activated receptor-&ggr; in smooth muscle cells (SMPG KO mice), we uncovered a complete absence of PVAT surrounding the vasculature, likely caused by peroxisome proliferator–activated receptor-&ggr; deletion in the perivascular adipocyte precursor cells as well. Lack of PVAT, which results in loss of its thermogenic activity, impaired vascular homeostasis, which caused temperature loss and endothelial dysfunction. We further showed that cold exposure inhibits atherosclerosis and improves endothelial function in mice with intact PVAT but not in SMPG KO mice as a result of impaired lipid clearance. Proinflammatory cytokine expression in PVAT is not altered on exposure to cold. Finally, prostacyclin released from PVAT contributes to the vascular protection against endothelial dysfunction. Conclusions— PVAT is a vasoactive organ with functional characteristics similar to BAT and is essential for intravascular thermoregulation of cold acclimation. This thermogenic capacity of PVAT plays an important protective role in the pathogenesis of atherosclerosis.

Peroxisome Proliferator-Activated Receptor δ Regulation of miR-15a in Ischemia-Induced Cerebral Vascular Endothelial Injury

Cerebral vascular endothelial cell (CEC) degeneration significantly contributes to blood–brain barrier (BBB) breakdown and neuronal loss after cerebral ischemia. Recently, emerging data suggest that peroxisome proliferator-activated receptor δ (PPARδ) activation has a potential neuroprotective role in ischemic stroke. Here we report for the first time that PPARδ is significantly reduced in oxygen-glucose deprivation (OGD)-induced mouse CEC death. Interestingly, PPARδ overexpression can suppress OGD-induced caspase-3 activity, Golgi fragmentation, and CEC death through an increase of bcl-2 protein levels without change of bcl-2 mRNA levels. To explore the molecular mechanisms, we have identified that upregulation of PPARδ can alleviate ODG-activated microRNA-15a (miR-15a) expression in CECs. Moreover, we have demonstrated that bcl-2 is a translationally repressed target of miR-15a. Intriguingly, gain- or loss-of-miR-15a function can significantly reduce or increase OGD-induced CEC death, respectively. Furthermore, we have identified that miR-15a is a transcriptional target of PPARδ. Consistent with the in vitro findings, we found that intracerebroventricular infusion of a specific PPARδ agonist, GW 501516 (2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid), significantly reduced ischemia-induced miR-15a expression, increased bcl-2 protein levels, and attenuated caspase-3 activity and subsequent DNA fragmentation in isolated cerebral microvessels, leading to decreased BBB disruption and reduced cerebral infarction in mice after transient focal cerebral ischemia. Together, these results suggest that PPARδ plays a vascular-protective role in ischemia-like insults via transcriptional repression of miR-15a, resulting in subsequent release of its posttranscriptional inhibition of bcl-2. Thus, regulation of PPARδ-mediated miR-15a inhibition of bcl-2 could provide a novel therapeutic strategy for the treatment of stroke-related vascular dysfunction.

Nitro-Oleic Acid Inhibits Angiotensin II–Induced Hypertension

Rationale Nitro-oleic acid (OA-NO2) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant vasculoprotective properties in vivo. OA-NO2 exerts cell signaling actions as a result of its strong electrophilic nature and mediates pleiotropic cell responses in the vasculature. Objective The present study sought to investigate the protective role of OA-NO2 in angiotensin (Ang) II–induced hypertension. Methods and Results We show that systemic administration of OA-NO2 results in a sustained reduction of Ang II–induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting hypertension established by Ang II infusion. OA-NO2 significantly inhibits Ang II contractile response as compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type 1 receptor (AT1R)-mediated signaling because vascular contraction by other G-protein–coupled receptors is not altered in response to OA-NO2 treatment. From the mechanistic viewpoint, OA-NO2 lowers Ang II–induced hypertension independently of peroxisome proliferation-activated receptor (PPAR)&ggr; activation. Rather, OA-NO2, but not OA, specifically binds to the AT1R, reduces heterotrimeric G-protein coupling, and inhibits IP3 (inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor. Conclusions These results demonstrate that OA-NO2 diminishes the pressor response to Ang II and inhibits AT1R-dependent vasoconstriction, revealing OA-NO2 as a novel antagonist of Ang II–induced hypertension.

Vascular Endothelial Cell-specific MicroRNA-15a Inhibits Angiogenesis in Hindlimb Ischemia

Background: MicroRNAs mediate angiogenesis in both physiological and pathological conditions, but underlying molecular mechanisms are largely unexplored. Results: Endothelial miR-15a negatively regulates angiogenesis in vivo and in vitro by suppression of FGF2 and VEGF. Conclusion: MiR-15a inhibits endothelial autonomous angiogenesis. Significance: MiR-15a is a negative regulator of angiogenesis and a potential target for the restorative therapy of ischemic diseases. The effects and potential mechanisms of the vascular endothelial cell (EC)-enriched microRNA-15a (miR-15a) on angiogenesis remain unclear. Here, we show a novel finding that EC-selective miR-15a transgenic overexpression leads to reduced blood vessel formation and local blood flow perfusion in mouse hindlimbs at 1–3 weeks after hindlimb ischemia. Mechanistically, gain- or loss-of-miR-15a function by lentiviral infection in ECs significantly reduces or increases tube formation, cell migration, and cell differentiation, respectively. By FGF2 and VEGF 3′-UTR luciferase reporter assays, Real-time PCR, and immunoassays, we further identified that the miR-15a directly targets FGF2 and VEGF to facilitate its anti-angiogenic effects. Our data suggest that the miR-15a in ECs can significantly suppress cell-autonomous angiogenesis through direct inhibition of endogenous endothelial FGF2 and VEGF activities. Pharmacological modulation of miR-15a function may provide a new therapeutic strategy to intervene against angiogenesis in a variety of pathological conditions.

Vascular Smooth Muscle Cell–Selective Peroxisome Proliferator–Activated Receptor-γ Deletion Leads to Hypotension

Background— Peroxisome proliferator–activated receptor-γ (PPARγ) agonists are commonly used to treat diabetes, although their PPARγ-dependent effects transcend their role as insulin sensitizers. Thiazolidinediones lower blood pressure (BP) in diabetic patients, whereas results from conventional/tissue-specific PPARγ experimental models suggest an important pleiotropic role for PPARγ in BP control. Little evidence is available on the molecular mechanisms underlying the role of vascular smooth muscle cell–specific PPARγ in basal vascular tone. Methods and Results— We show that vascular smooth muscle cell–selective deletion of PPARγ impairs vasoactivity with an overall reduction in BP. Aortic contraction in response to norepinephrine is reduced and vasorelaxation is enhanced in response to β-adrenergic receptor (β-AdR) agonists in vitro. Similarly, vascular smooth muscle cell–selective PPARγ knockout mice display a biphasic response to norepinephrine in BP, reversible on administration of β-AdR blocker, and enhanced BP reduction on treatment with β-AdR agonists. Consistent with enhanced β2-AdR responsiveness, we found that the absence of PPARγ in vascular smooth muscle cells increased β2-AdR expression, possibly leading to the hypotensive phenotype during the rest phase. Conclusion— These data uncovered the β2-AdR as a novel target of PPARγ transcriptional repression in vascular smooth muscle cells and indicate that PPARγ regulation of β2-adrenergic signaling is important in the modulation of BP.

Altered long non-coding RNA transcriptomic profiles in brain microvascular endothelium after cerebral ischemia

The brain endothelium is an important therapeutic target for the inhibition of cerebrovascular dysfunction in ischemic stroke. Previously, we documented the important regulatory roles of microRNAs in the cerebral vasculature, in particular the cerebral vascular endothelium. However, the functional significance and molecular mechanisms of other classes of non-coding RNAs in the regulation of cerebrovascular endothelial pathophysiology after stroke are completely unknown. Using RNA sequencing (RNA-seq) technology, we profiled long non-coding RNA (lncRNA) expressional signatures in primary brain microvascular endothelial cells (BMECs) after oxygen-glucose deprivation (OGD), an in vitro mimic of ischemic stroke conditions. After 16h of OGD exposure, the expression levels for 362 of the 10,677 lncRNAs analyzed changed significantly, including a total of 147 lncRNAs increased and 70 lncRNAs decreased by more than 2-fold. Among them, the most highly upregulated lncRNAs include Snhg12, Malat1, and lnc-OGD 1006, whereas the most highly downregulated lncRNAs include 281008D09Rik, Peg13, and lnc-OGD 3916. Alteration of the most highly upregulated/downregulated ODG-responsive lncRNAs was further confirmed in cultured BMECs after OGD as well as isolated cerebral microvessels in mice following transient middle cerebral artery occlusion (MCAO) and 24h reperfusion by the quantitative real-time PCR approach. Moreover, promoter analysis of altered ODG-responsive endothelial lncRNA genes by bioinformatics showed substantial transcription factor binding sites on lncRNAs, implying potential transcriptional regulation of those lncRNAs. These findings are the first to identify OGD-responsive brain endothelial lncRNAs, which suggest potential pathological roles for these lncRNAs in mediating endothelial responses to ischemic stimuli. Endothelial-selective lncRNAs may function as a class of novel master regulators in cerebrovascular endothelial pathologies after ischemic stroke.

Angiogenesis-regulating microRNAs and Ischemic Stroke

Stroke is a leading cause of death and disability worldwide. Ischemic stroke is the dominant subtype of stroke and results from focal cerebral ischemia due to occlusion of major cerebral arteries. Thus, the restoration or improvement of reduced regional cerebral blood supply in a timely manner is very critical for improving stroke outcomes and poststroke functional recovery. The recovery from ischemic stroke largely relies on appropriate restoration of blood flow via angiogenesis. Newly formed vessels would allow increased cerebral blood flow, thus increasing the amount of oxygen and nutrients delivered to affected brain tissue. Angiogenesis is strictly controlled by many key angiogenic factors in the central nervous system, and these molecules have been well-documented to play an important role in the development of angiogenesis in response to various pathological conditions. Promoting angiogenesis via various approaches that target angiogenic factors appears to be a useful treatment for experimental ischemic stroke. Most recently, microRNAs (miRs) have been identified as negative regulators of gene expression in a post-transcriptional manner. Accumulating studies have demonstrated that miRs are essential determinants of vascular endothelial cell biology/angiogenesis as well as contributors to stroke pathogenesis. In this review, we summarize the knowledge of stroke-associated angiogenic modulators, as well as the role and molecular mechanisms of stroke-associated miRs with a focus on angiogenesis-regulating miRs. Moreover, we further discuss their potential impact on miR-based therapeutics in stroke through targeting and enhancing post-ischemic angiogenesis.

Non-coding RNAs in cerebral endothelial pathophysiology: emerging roles in stroke.

Cerebral vascular endothelial cells form the major element of the blood-brain barrier (BBB) and constitute the primary interface between circulating blood and brain parenchyma. The structural and functional changes in cerebral endothelium during cerebral ischemia are well known to result in BBB disruption, vascular inflammation, edema, and angiogenesis. These complex pathological processes directly contribute to brain infarction, neurological deficits, and post-stroke neurovascular remodeling. Ischemic endothelial dysfunction appears to be tightly controlled by multiple gene signaling networks. Non-coding RNAs (ncRNAs) are functional RNA molecules that are generally not translated into proteins but can actively regulate the expression and function of many thousands of protein-coding genes by different mechanisms. Various classes of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small nucleolar RNAs (snoRNAs) and piwi-interacting RNAs (piRNAs), are highly expressed in the cerebrovascular endothelium where they serve as critical mediators to maintain normal cerebral vascular functions. Dysregulation of ncRNA activities has been closely linked to the pathophysiology of cerebral vascular endothelium and neurologic functional disorders in the brains response to ischemic stimuli. In this review, we summarize recent advancements of these ncRNA mediators in the brain vasculature, highlighting the specific roles of endothelial miRNAs in stroke.

KLF11 mediates PPARγ cerebrovascular protection in ischaemic stroke

Peroxisome proliferator-activated receptor gamma (PPARγ) is emerging as a major regulator in neurological diseases. However, the role of (PPARγ) and its co-regulators in cerebrovascular endothelial dysfunction after stroke is unclear. Here, we have demonstrated that (PPARγ) activation by pioglitazone significantly inhibited both oxygen-glucose deprivation-induced cerebral vascular endothelial cell death and middle cerebral artery occlusion-triggered cerebrovascular damage. Consistent with this finding, selective (PPARγ) genetic deletion in vascular endothelial cells resulted in increased cerebrovascular permeability and brain infarction in mice after focal ischaemia. Moreover, we screened for (PPARγ) co-regulators using a genome-wide and high-throughput co-activation system and revealed KLF11 as a novel (PPARγ) co-regulator, which interacted with (PPARγ) and regulated its function in mouse cerebral vascular endothelial cell cultures. Interestingly, KLF11 was also found as a direct transcriptional target of (PPARγ). Furthermore, KLF11 genetic deficiency effectively abolished pioglitazone cytoprotection in mouse cerebral vascular endothelial cell cultures after oxygen-glucose deprivation, as well as pioglitazone-mediated cerebrovascular protection in a mouse middle cerebral artery occlusion model. Mechanistically, we demonstrated that KLF11 enhanced (PPARγ) transcriptional suppression of the pro-apoptotic microRNA-15a (miR-15a) gene, resulting in endothelial protection in cerebral vascular endothelial cell cultures and cerebral microvasculature after ischaemic stimuli. Taken together, our data demonstrate that recruitment of KLF11 as a novel (PPARγ) co-regulator plays a critical role in the cerebrovascular protection after ischaemic insults. It is anticipated that elucidating the coordinated actions of KLF11 and (PPARγ) will provide new insights into understanding the molecular mechanisms underlying (PPARγ) function in the cerebral vasculature and help to develop a novel therapeutic strategy for the treatment of stroke.

Vascular PPARδ Protects Against Stroke-Induced Brain Injury

Objective—To investigate the effects of peroxisome proliferator-activated receptor (PPAR)&dgr; in the cerebral vasculature following stroke-induced brain injury. Methods and Results—Here, we report a novel finding that selective PPAR&dgr; genetic deletion in vascular smooth muscle cells (VSMCs) resulted in increased cerebrovascular permeability and brain infarction in mice after middle cerebral artery occlusion (MCAO). Mechanistically, we revealed for the first time that PPAR&dgr; expression is reduced, but matrix metalloproteinase (MMP)-9 activity is increased in cultured VSMCs after oxygen–glucose deprivation and also in the cerebral cortex of mice following MCAO. Moreover, gain- and loss of PPAR&dgr; function in VSMCs significantly reduces and increases oxygen–glucose deprivation–induced MMP-9 activity, respectively. We have further identified that MMP-9 is a direct target of PPAR&dgr;-mediated transrepression by chromatin immunoprecipitation and PPAR&dgr; transcriptional activity assays. Furthermore, inhibition of MMP-9 activity by lentiviral MMP-9 short hairpin RNA effectively improves cerebrovascular permeability and reduces brain infarction in VSMC-selective PPAR&dgr; conditional knockout mice after MCAO. Conclusion—Our data demonstrate that PPAR&dgr; in VSMCs can prevent ischemic brain injury by inhibition of MMP-9 activation and attenuation of postischemic inflammation. The pharmacological activation of PPAR&dgr; may provide a new therapeutic strategy to treat stroke-induced vascular and neuronal damage.