Noninvasive disruption of the blood-brain barrier in the marmoset monkey.

The common marmoset monkey (Callithrix jacchus) is a species of rising prominence in the neurosciences due to its small size, ease of handling, fast breeding, and its shared functional and structural brain characteristics with Old World primates. With increasing attention on modeling human brain diseases in marmosets, understanding how to deliver therapeutic or neurotropic agents to the marmoset brain noninvasively is of great preclinical importance. In other species, including humans, transcranial focused ultrasound (tFUS) aided by intravenously injected microbubbles has proven to be a transient, reliable, and safe method for disrupting the blood-brain barrier (BBB), allowing the focal passage of therapeutic agents that do not otherwise readily traverse the tight endothelial junctions of the BBB. The critical gap that we address here is to document parameters to disrupt the BBB reliably and safely in marmosets using tFUS. By integrating our marmoset brain atlases and the use of a marmoset-specific stereotactic targeting system, we conduct a series of systematic transcranial sonication experiments in nine marmosets. We demonstrate the effects of center frequency, acoustic pressure, burst period, and duration, establish a minimum microbubble dose, estimate microbubble clearance time, and estimate the duration that the BBB remains open to passage. Successful BBB disruption is reported in vivo with MRI-based contrast agents, as well as Evans blue staining assessed ex vivo. Histology (Hematoxylin and Eosin staining) and immunohistochemistry indicate that the BBB can be safely and reliably opened with the parameters derived from these experiments. The series of experiments presented here establish methods for safely, reproducibly, and focally perturbing the BBB using tFUS in the common marmoset monkey that can serve as a basis for noninvasive delivery of therapeutic or neurotropic agents.

SUMOylation promotes survival and integration of neural stem cell grafts in ischemic stroke.

BACKGROUND: Neural stem cell (NSC)-based therapies hold great promise for treating diseases of the central nervous system (CNS). However, several fundamental problems still need to be overcome to fully exploit the clinical potential of NSC therapeutics. Chief among them is the limited survival of NSC grafts within hostile microenvironments. METHODS: Herein, we sought to engineer NSCs in an effort to increase graft survival within ischemic brain lesions via upregulation of global SUMOylation, a post-translational modification critically involved in mediating tolerance to ischemia/reperfusion. FINDINGS: NSCs overexpressing the SUMO E2-conjugase Ubc9 displayed resistance to oxygen-glucose-deprivation/restoration of oxygen/glucose (OGD/ROG) and enhanced neuronal differentiation in vitro, as well as increased survival and neuronal differentiation when transplanted in mice with transient middle cerebral artery occlusion in vivo. INTERPRETATION: Our work highlights a critical role for SUMOylation in NSC biology and identifies a biological pathway that can be targeted to increase the effectiveness of exogenous stem cell medicines in ischemic stroke. FUND: Intramural Research Program of the NINDS/NIH, the Italian Multiple Sclerosis Foundation (FISM), the Bascule Charitable Trust, NIH-IRTA-OxCam and Wellcome Trust Research Training Fellowships.

Neuroprotective Effects of MAGL (Monoacylglycerol Lipase) Inhibitors in Experimental Ischemic Stroke.

BACKGROUND AND PURPOSE: MAGL (monoacylglycerol lipase) is an enzyme that hydrolyzes the endocannabinoid 2-arachidonoylglycerol and regulates the production of arachidonic acid and prostaglandins-substances that mediate tissue inflammatory response. Here, we have studied the effects of the selective MAGL inhibitors JZL184 and MJN110 and their underlying molecular mechanisms on 3 different experimental models of focal cerebral ischemia. METHODS: SHR (spontaneously hypertensive rats) and normotensive WKY (Wistar Kyoto) rats were subject to an intracortical injection of the potent vasoconstrictor endothelin-1, permanent occlusion of a distal segment of the middle cerebral artery via craniectomy, or transient occlusion of the middle cerebral artery by the intraluminal suture method. JZL184 or MJN110 was administered 60 minutes after focal cerebral ischemia. Infarct volumes, hemispheric swelling, and functional outcomes were assessed between days 1 to 28 by magnetic resonance imaging, histology, and behavioral tests. RESULTS: Pharmacological inhibition of MAGL significantly attenuated infarct volume and hemispheric swelling. MAGL inhibition also ameliorated sensorimotor deficits, suppressed inflammatory response, and decreased the number of degenerating neurons. These beneficial effects of MAGL inhibition were not fully abrogated by selective antagonists of cannabinoid receptors, indicating that the anti-inflammatory effects are caused by inhibition of eicosanoid production rather than by activation of cannabinoid receptors. CONCLUSIONS: Our results suggest that MAGL may contribute to the pathophysiology of focal cerebral ischemia and is thus a promising therapeutic target for the treatment of ischemic stroke.

Synthetic Oligodeoxynucleotides Containing Multiple Telemeric TTAGGG Motifs Suppress Inflammasome Activity in Macrophages Subjected to Oxygen and Glucose Deprivation and Reduce Ischemic Brain Injury in Stroke-Prone Spontaneously Hypertensive Rats.

The immune system plays a fundamental role in both the development and pathobiology of stroke. Inflammasomes are multiprotein complexes that have come to be recognized as critical players in the inflammation that ultimately contributes to stroke severity. Inflammasomes recognize microbial and host-derived danger signals and activate caspase-1, which in turn controls the production of the pro-inflammatory cytokine IL-1ß. We have shown that A151, a synthetic oligodeoxynucleotide containing multiple telemeric TTAGGG motifs, reduces IL-1ß production by activated bone marrow derived macrophages that have been subjected to oxygen-glucose deprivation and LPS stimulation. Further, we demonstrate that A151 reduces the maturation of caspase-1 and IL-1ß, the levels of both the iNOS and NLRP3 proteins, and the depolarization of mitochondrial membrane potential within such cells. In addition, we have demonstrated that A151 reduces ischemic brain damage and NLRP3 mRNA levels in SHR-SP rats that have undergone permanent middle cerebral artery occlusion. These findings clearly suggest that the modulation of inflammasome activity via A151 may contribute to a reduction in pro-inflammatory cytokine production by macrophages subjected to conditions that model brain ischemia and modulate ischemic brain damage in an animal model of stroke. Therefore, modulation of ischemic pathobiology by A151 may have a role in the development of novel stroke prevention and therapeutic strategies.

Global SUMOylation is a molecular mechanism underlying hypothermia-induced ischemic tolerance.

The molecular mechanisms underlying hypothermic neuroprotection have yet to be fully elucidated. Herein we demonstrate that global SUMOylation, a form of post-translational modification with the Small Ubiquitin-like MOdifer, participates in the multimodal molecular induction of hypothermia-induced ischemic tolerance. Mild (32°C) to moderate (28°C) hypothermic treatment(s) during OGD (oxygen-glucose-deprivation) or ROG (restoration of oxygen/glucose) increased global SUMO-conjugation levels and protected cells (both SHSY5Y and E18 rat cortical neurons) from OGD and ROG-induced cell death. Hypothermic exposure either before or after permanent middle cerebral artery occlusion (pMCAO) surgery in wild type mice increased global SUMO-conjugation levels in the brain and in so doing protected these animals from pMCAO-induced ischemic damage. Of note, hypothermic exposure did not provide an additional increase in protection from pMCAO-induced ischemic brain damage in Ubc9 transgenic (Ubc9 Tg) mice, which overexpress the sole E2 SUMO conjugating enzyme and thereby display elevated basal levels of global SUMOylation under normothermic conditions. Such evidence suggests that increases in global SUMOylation are critical and may account for a substantial part of the observed increase in cellular tolerance to brain ischemia caused via hypothermia.

A new role for downstream Toll-like receptor signaling in mediating immediate early gene expression during focal cerebral ischemia.

To better understand the role of downstream Toll-like receptor (TLR) signaling during acute cerebral ischemia, we performed cDNA microarrays, on brain RNA, and cytokine arrays, on serum, from wild type (WT), MyD88-/- and TRIF-mutant mice, at baseline and following permanent middle cerebral artery occlusion (pMCAO). The acute stress response pathway was among the top pathways identified by Ingenuity Pathway Analysis of microarray data. We used real-time polymerase chain reaction to confirm the expression of four immediate early genes; EGR1, EGR2, ARC, Nurr77, in this pathway, and insulin degrading enzyme (IDE). Compared to WT, baseline immediate early gene expression was increased up to10-fold in MyD88-/- and TRIF-mutant mice. However, following pMCAO, immediate early gene expression remained unchanged, from this elevated baseline in these mice, but increased up to 12-fold in WT. Furthermore, expression of IDE, which also degrades ß-amyloid, decreased significantly only in TRIF-mutant mice. Finally, sE-Selectin, sICAM, sVCAM-1, and MMP-9 levels were significantly decreased only in MyD88-/- compared with WT mice. We thus report a new role for downstream TLR signaling in immediate early gene expression during acute cerebral ischemia. We also show that the TRIF pathway regulates IDE expression; a major enzyme that clears ß-amyloid from the brain.

A method for hypothermia-induction and maintenance allows precise body and brain temperature control in mice.

The benefits as well as mechanisms of hypothermia in brain injuries are actively studied at the bench and in the clinic. However, methods used in controlling hypothermia vary among laboratories, and usually brain temperatures are not monitored directly in animals due to the need for an invasive procedure. Here we show a method, water immersion technique, which we developed recently to regulate body temperature in mice during hypothermia process. This method significantly reduced the temperature variation around target temperature. Importantly, this method demonstrated a parallel and consistent relationship between rectal temperature and brain temperature (the brain temperature was consistently 0.5C higher than rectal temperature) throughout hypothermia maintenance. This technique may be well adapted to hypothermia studies in mice and other rodents, especially to the assessment and regulation of brain temperature during studies.

Downstream Toll-like receptor signaling mediates adaptor-specific cytokine expression following focal cerebral ischemia.

BACKGROUND: Deletion of some Toll-like receptors (TLRs) affords protection against cerebral ischemia, but disruption of their known major downstream adaptors does not. To determine whether compensation in the production of downstream effectors by one pathway when the other is disrupted can explain these findings, we examined cytokine/chemokine expression and inflammatory infiltrates in wild-type (WT), MyD88(-/-) and TRIF-mutant mice following permanent middle cerebral artery occlusion (pMCAO). METHODS: Cytokine/chemokine expression was measured with a 25-plex bead array in the serum and brains of all three groups of mice at baseline (no surgery/naïve) and at 3 hours and 24 hours following pMCAO. Brain inflammatory and neutrophil infiltrates were examined 24 hours following pMCAO. RESULTS: IL-6, keratinocyte chemoattractant (KC), granulocyte colony-stimulating factor (G-CSF) and IL-10 were significantly decreased in MyD88(-/-) mice compared to WT mice following pMCAO. Significantly, decreased levels of the neutrophil chemoattractants KC and G-CSF corresponded with a trend toward fewer neutrophils in the brains of MyD88(-/-) mice. IP-10 was significantly decreased when either pathway was disrupted. MIP-1 α was significantly decreased in TRIF-mutant mice, consistent with TRIF-dependent production. MyD88(-/-) mice showed elevations of a number of Th2 cytokines, such as IL-13, at baseline, which became significantly decreased following pMCAO. CONCLUSIONS: Both MyD88 and TRIF mediate pathway-specific cytokine production following focal cerebral ischemia. Our results also suggest a compensatory Th2-type skew at baseline in MyD88(-/-) mice and a paradoxical switch to a Th1 phenotype following focal cerebral ischemia. The MyD88 pathway directs the expression of neutrophil chemoattractants following cerebral ischemia.

Elevated global SUMOylation in Ubc9 transgenic mice protects their brains against focal cerebral ischemic damage.

We have previously shown that a massive increase in global SUMOylation occurs during torpor in ground squirrels, and that overexpression of Ubc9 and/or SUMO-1 in cell lines and cortical neurons protects against oxygen and glucose deprivation. To examine whether increased global SUMOylation protects against ischemic brain damage, we have generated transgenic mice in which Ubc9 is expressed strongly in all tissues under the chicken ß-actin promoter. Ubc9 expression levels in 10 founder lines ranged from 2 to 30 times the endogenous level, and lines that expressed Ubc9 at modestly increased levels showed robust resistance to brain ischemia compared to wild type mice. The infarction size was inversely correlated with the Ubc9 expression levels for up to five times the endogenous level. Although further increases showed no additional benefit, the Ubc9 expression level was highly correlated with global SUMO-1 conjugation levels (and SUMO-2,3 levels to a lesser extent) up to a five-fold Ubc9 increase. Most importantly, there were striking reciprocal relationships between SUMO-1 (and SUMO-2,3) conjugation levels and cerebral infarction volumes among all tested animals, suggesting that the limit in cytoprotection by global SUMOylation remains undefined. These results support efforts to further augment global protein SUMOylation in brain ischemia.

Disruption of downstream MyD88 or TRIF Toll-like receptor signaling does not protect against cerebral ischemia.

Toll-like receptor (TLR) signaling plays an important role in cerebral ischemia, but downstream signaling events, which can be organ-specific, are incompletely understood. We thereby investigated involvement of the MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling pathways in 2 in vitro and 2 in vivo models of cerebral ischemia. For in vitro studies, we used a model of oxygen-glucose deprivation (OGD) followed by flow cytometric analysis to determine:1) viability of PC12 cells following knock-down with MyD88 siRNA compared to negative control siRNA and 2) viability, apoptosis and necrosis of cortical neurons from MyD88 null (-/-) , TRIF mutant, and wild type (WT) mice. In addition, in vivo, 1) We examined CA1 neuronal survival 7 days after global forebrain ischemia and 2) infarct volumes 24h after Middle Cerebral Artery Occlusion (MCAO) in all 3 types of mice. OGD: 1) There were no differences in either percent viability of PC12 cells transfected with MyD88 compared to negative control siRNA or 2) in percent viability, apoptosis and necrosis of cortical neurons from MyD88-/-,TRIF mutant and WT mice. Global ischemia: neuronal survival was similar in all 3 groups of mice. Finally, MCAO: infarct volumes were not statistically different among all 3 groups of mice: MyD88-/-, 23.9±9.9 mm(3), TRIF mutant, 26.7±5.8 mm(3) and WT, 17.9±8.4mm(3). These findings show that disruption of MyD88 or TRIF signaling does not confer protection in brain ischemia and suggests the possibility of additional or alternate downstream adaptors during TLR signaling in cerebral ischemia.

Mucosal tolerance to E-selectin promotes the survival of newly generated neuroblasts via regulatory T-cell induction after stroke in spontaneously hypertensive rats.

Neuroblasts in the subventricular zone (SVZ) proliferate markedly after brain ischemia, and migrate to the site of injury along with blood vessels. However, a large fraction of stroke-generated neuroblasts die shortly after being born, in part, because of local inflammation. In spontaneously hypertensive rats (SHRs) subjected to permanent middle cerebral artery occlusion, we primed E-selectin-specific regulatory T cells (Tregs) by repetitive intranasal administration of recombinant E-selectin to target local secretion of immunomodulating, antiinflammatory cytokines to activating blood vessel segments. E-selectin-tolerized SHRs had decreased infarction volumes, and increased numbers of Tregs in the cervical lymph nodes and ischemic brain. The brain Tregs were distributed primarily in periinfarct regions. E-selectin tolerization did not alter cellular proliferation in the ipsilateral SVZ after stroke, but the expression of tumor necrosis factor on vascular niche blood vessels was suppressed and both doublecortin protein levels and the number of newly generated neuroblasts or neurons were increased in the brain. This enhanced survival of neural progenitor cells and neurons was paralleled by improved functional performance. These studies suggest that E-selectin-specific Tregs can modulate the efficacy of neurogenesis after ischemia and promote repair after brain injury.

Transplantation of human undifferentiated embryonic stem cells into a myocardial infarction rat model.

Human embryonic stem (hES) cells hold great therapeutic potential for cell transplantation. To date, it remains uncertain whether undifferentiated hES cells can differentiate into cardiac lineage in vivo during myocardial infarction. Here we provide the first report that undifferentiated hES cells can survive in rat hearts during myocardial infarction without the formation of teratoma using undifferentiated green fluorescent protein (GFP)-transgenic hES cells. Using a laser-capture microscope to dissect the GFP-positive cell area from the hES-injected hearts, we documented the expression of human cardiac-specific genes, including GATA-4, Nkx-2.5, and cardiac troponin I. Taken together, our results demonstrate that undifferentiated hES cells can be driven to the cardiac lineage under the local injured environment in the heart, which may provide a potential method for regenerating de novo myocardium to treat myocardial infarction.

Neuronal chemorepellent Slit2 inhibits vascular smooth muscle cell migration by suppressing small GTPase Rac1 activation.

The Slits are secreted proteins with roles in axonal guidance and leukocyte migration. On binding to Robo receptors, Slit2 repels developing axons and inhibits leukocyte chemotaxis. Slit2 is cleaved into Slit2-N, a protein tightly binding to cell membranes, and Slit2-C, a diffusible fragment. In the present study, we characterized the functional role of Slit2-N in vascular smooth muscle cells (VSMCs) and the cell association properties of 2 truncated versions of Slit2-N. Here, we document for the first time that Slit2-N is a chemorepellent of VSMCs. Intact blood vessels expressed Slit2 and Robo receptors as demonstrated by immunohistochemistry and quantitative real time PCR. Recombinant Slit2-N prevented the platelet-derived growth factor (PDGF)-stimulated migration of VSMCs. Slit2-N also abrogated PDGF-mediated activation of small guanosine triphosphatase (GTPase) Rac1, a member of the Rho GTPase superfamily of proteins involved in regulating the actin cytoskeleton. Furthermore, Slit2-N inhibited the PDGF-induced formation of lamellipodia, a crucial cytoskeletal reorganization event for cell motility. Slit2-N had no effect on the PDGF-mediated increase in DNA synthesis determined by [3H]thymidine uptake, suggesting that VSMC growth is unaffected by Slit2. Analysis of 2 engineered Slit2-N fragments (Slit2-N/1118 and Slit2-N/1121) indicated that 3 amino acids upstream of the putative cleavage site (Arg1121, Thr1122) are involved in the association of Slit2-N to the cell membrane. Our data assign a novel functional role to Slit2 in vascular function and show that cell guidance mechanisms that operate in the developing central nervous system are conserved in VSMCs.

Impaired expression of PPAR gamma protein contributes to the exaggerated growth of vascular smooth muscle cells in spontaneously hypertensive rats.

Peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor family, has been implicated in the regulation of vascular smooth muscle cell (VSMC) growth; however, the underlying mechanisms are still not fully understood. We hypothesized that PPAR gamma functional deficiency may contribute to the enhanced proliferation of VSMC associated with hypertension in spontaneously hypertensive rats (SHR). We observed that PPAR gamma mRNA level in SHR VSMC was 3 approximately 4 fold higher than that from Wistar-Kyoto rats (WKY), but the protein expression levels of PPAR gamma are significantly lower in SHR than WKY VSMC, suggesting an impaired control of PPAR gamma protein expression in SHR VSMC. The deficiency of PPAR gamma protein expression in SHR VSMC was demonstrated by PPAR gamma reporter gene assays. Furthermore, the exaggerated growth of SHR VSMC was markedly attenuated by adenoviral PPAR gamma overexpression. Taken together, our results provided the first direct evidence that impaired expression of PPAR gamma protein contributes to the exaggerated growth of SHR VSMC.

Rad GTPase attenuates vascular lesion formation by inhibition of vascular smooth muscle cell migration.

BACKGROUND: Rad (Ras associated with diabetes) GTPase is a prototypic member of a new subfamily of Ras-related GTPases with unique structural features, although its physiological role remains largely unknown. In the present study, we characterized the Rad function in vascular smooth muscle cells (VSMCs) and the influence of adenovirus-mediated Rad (Ad-Rad) gene delivery on vascular remodeling after experimental angioplasty. METHODS AND RESULTS: We documented for the first time that neointimal formation using balloon-injured rat carotid arteries was associated with a significant increase in Rad expression as determined by immunohistochemistry and quantitative real-time reverse-transcriptase polymerase chain reaction. The levels of Rad expression in VSMCs were highly induced by platelet-derived growth factor and tumor necrosis factor-alpha. Morphometric analyses 14 days after injury revealed significantly diminished neointimal formation in the Ad-Rad-treated carotid arteries compared with Ad-GFP or PBS controls, whereas the mutated form of Rad GTPase, which can bind GDP but not GTP, increased neointimal formation. Overexpression of Rad significantly inhibited the attachment and migration of VSMCs. In addition, Rad expression dramatically reduced the formation of focal contacts and stress fibers in VSMCs by blocking the Rho/ROK signaling pathway. CONCLUSIONS: Our data clearly identified Rad GTPase as a novel and critical mediator that inhibits vascular lesion formation. Manipulation of the Rad signaling pathway may provide new therapeutic approaches that will limit vascular pathological remodeling.

Coordinate Notch3-hairy-related transcription factor pathway regulation in response to arterial injury. Mediator role of platelet-derived growth factor and ERK.

The Notch family of receptors and downstream effectors plays a critical role in cell fate determination during vascular ontogeny. Moreover, the human cerebral autosomal dominant artriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) syndrome of premature stroke and dementia is a heritable arteriopathy with alterations in vascular smooth muscle cells (VSMCs) resulting from mutations within Notch3. However, the expression and regulation of the Notch and hairy-related transcription factor (HRT) pathway in adult VSMCs in vitro and in vivo remain poorly characterized. The present study documents that the well-described modulation of VSMC fate in response to vascular injury and growth factor activation involves a coordinate regulation of the Notch and HRT pathways. Furthermore, platelet-derived growth factor promotes a similar coordinate down-regulation of the Notch receptors and HRT genes in cultured VSMCs via an ERK-dependent signaling pathway. Moreover, we established that HRT1 and HRT2 are direct downstream target genes of Notch3 signaling in VSMCs and determined that the activity of the nuclear protein RBP-Jk is essential for their regulation. These findings provide initial insight into the context- and cell type-dependent coordinate regulation of Notch3 and downstream HRT transcriptional pathway effector genes in VSMCs in vitro and in vivo that may have important implications for understanding the role of Notch signaling in human health and vascular disease.

Notch3 signaling in vascular smooth muscle cells induces c-FLIP expression via ERK/MAPK activation. Resistance to Fas ligand-induced apoptosis.

Mutations in the Notch3 receptor result in the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy (CADASIL) syndrome, a heritable arteriopathy predisposing to early onset stroke. Based upon clinical evidence that CADASIL arteriopathy results in degeneration and loss of vascular smooth muscle cells (VSMC) from the arterial wall, we postulated that Notch3 signaling is a critical determinant of VSMC survival. We initially established that both transient and constitutive Notch3 signaling promoted VSMC survival in response to the proapoptotic Fas ligand (FasL). Resistance to FasL-induced apoptosis was associated with the induction of c-FLIP, a primary inhibitor of the FasL signaling pathway. We determined that Notch3's regulation of c-FLIP was independent of the activity of the classical DNA-binding protein, RBP-Jk, but dependent upon cross-talk activation of the ERK/MAPK pathway. We extended our observations to the in vivo context by determining a coordinate regulation of Notch3 and c-FLIP within the arterial wall in response to injury. Furthermore, we defined that expression levels of Notch3 and c-FLIP are coordinately up-regulated within the neointima of remodeled arteries. Taken together, these findings provide initial evidence that Notch3 signaling may be a critical determinant of VSMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis via signaling cross-talk with the ERK/MAPK pathway.

A role for the beta-catenin/T-cell factor signaling cascade in vascular remodeling.

Beta-catenin and T cell factor (Tcf) are distal components of the highly conserved Wnt pathway that govern cell fate and proliferation in lower organisms. Thus, we hypothesized that the regulation of beta-catenin and Tcf played a critical role in vascular remodeling. The first objective was to define beta-catenin expression in vascular smooth muscle cells (VSMCs) after balloon injury. Indeed, beta-catenin mRNA and protein were significantly elevated 7 days after balloon injury in the rat carotid artery. We hypothesized that beta-catenin accumulation in response to vascular injury inhibited VSMC apoptosis. In line with our hypothesis, transfection of a degradation-resistant beta-catenin transgene into rat VSMCs significantly inhibited apoptosis. Accumulation of beta-catenin also resulted in a 10-fold increase in the activation of Tcf. To test if Tcf was necessary to confer beta-catenin-induced survival, loss of function studies were carried out with a dominant negative Tcf-4 transgene lacking the beta-catenin binding domain, Tcf4(N31). Indeed, loss of Tcf-4 activity abolished beta-catenin-induced survival. We further postulated that beta-catenin and Tcf promoted cell cycle progression by activating cyclin D1, a target gene of Tcf-4. Beta-catenin activated cyclin D1, and this activation was partially blocked with loss of Tcf-4. In parallel, blockade of Tcf-4 resulted in inhibition of [3H]thymidine incorporation and partial blockade of the G1-S phase transition. In conclusion, beta-catenin and Tcf-4 play a dual role in vascular remodeling by inhibiting VSMC apoptosis and promoting proliferation.

Peroxisome proliferator-activated receptor delta is up-regulated during vascular lesion formation and promotes post-confluent cell proliferation in vascular smooth muscle cells.

Although peroxisome proliferator-activated receptor (PPAR) delta is widely expressed in many tissues, the role of PPARdelta is poorly understood. In this study, we report that PPARdelta was up-regulated in vascular smooth muscle cells (VSMC) during vascular lesion formation. By using Northern blot analysis, we demonstrated that PPARdelta was increased by 3-4-fold in VSMC treated with platelet-derived growth factor (PDGF) (20 ng/ml). In addition, PDGF-induced PPARdelta mRNA expression neither needs de novo protein synthesis nor affects the stability of PPARdelta mRNA in VSMC. Preincubation of VSMC with phosphatidylinositol 3-kinase inhibitor (LY294002, 50 micromol/liter) or infection of VSMC with an adenovirus carrying the gene for a dominant negative form of Akt abrogated PDGF-induced PPARdelta mRNA expression, suggesting that phosphatidylinositol 3-kinase/Akt signaling pathway is involved in the regulation of PDGF-induced PPARdelta mRNA expression in VSMC. To explore the role of PPARdelta in VSMC, we generated rat vascular smooth muscle cells (A7r5) stably overexpressing PPARdelta and the control green fluorescent protein. Overexpression of PPARdelta in VSMC increased post-confluent cell proliferation by increasing the cyclin A and CDK2 as well as decreasing p57(kip2). Taken together, the results suggest that PPARdelta plays an important role in the pathology of diseases associated with VSMC proliferation, such as primary atherosclerosis and restenosis.