Michael M. Wang

Sex differences in cell death

Female patients experience substantial neuroprotection after experimental stroke compared with male patients, a finding attributed to the protective effects of gonadal hormones. This study examined the response of male‐ and female‐derived organotypic hippocampal slices to oxidative and excitotoxic injury. Both oxygen and glucose deprivation and N‐methyl‐D‐aspartic acid exposure led to neuronal death; however, female‐derived cultures sustained less injury than male‐derived cultures. Cell death after oxygen and glucose deprivation was ameliorated in male cultures, but not female cultures, by the addition of 7‐nitroindazole, a neuronal nitric oxide synthase inhibitor. These studies have relevance to researchers investigating neuroprotective agents in mixed sex experiments. Ann Neurol 2005

Estradiol Regulates Angiopoietin-1 mRNA Expression Through Estrogen Receptor-α in a Rodent Experimental Stroke Model

Background and Purpose— Female, compared with male, animals are protected from cerebral ischemic injury. Physiological concentrations of 17&bgr;-estradiol (E2) reduce damage in experimental stroke. E2 augments angiogenesis in reproductive organs and noncerebral vascular beds. We hypothesized that E2 protects brain in stroke through modulation of angiogenesis. We quantified molecular markers of angiogenesis and capillary density before and after unilateral middle cerebral artery occlusion (MCAO). Methods— Female animals were ovariectomized, treated with 25 &mgr;g E2 or placebo implants, and subjected to 2-hour MCAO and 22 hours of reperfusion. Brain angiopoietin-1 (Ang-1), Ang-2, Tie-1, Tie-2, vascular endothelial growth factor (VEGF), VEGF R1, and VEGF R2 mRNA levels were determined by RNAse protection assays, and CD31-positive vessels were counted. Results— E2, but not ischemia, upregulated cerebral Ang-1 mRNA by 49%. Capillary density was higher in the brains of E2-treated animals. In estrogen receptor-&agr; knockout (ERKO) mice, E2-mediated induction of Ang-1 mRNA was absent relative to wild-type littermates. Conclusions— These results suggest that E2 increases Ang-1 and enhances capillary density in brain under basal conditions, priming the MCA territory for survival after experimental focal ischemia.

Myeloid-Specific Deletion of the Mineralocorticoid Receptor Reduces Infarct Volume and Alters Inflammation During Cerebral Ischemia

Background and Purpose— Mineralocorticoid receptor (MR) antagonists have protective effects in rodent models of ischemic stroke, but the cell type-specific actions of these drugs are unknown. In the present study, we examined the contribution of myeloid cell MR during focal cerebral ischemia using myeloid-specific MR knockout mice. Methods— Myeloid-specific MR knockout mice were subjected to transient (90 minutes) middle cerebral artery occlusion followed by 24 hours reperfusion (n=5 to 7 per group). Ischemic cerebral infarcts were identified by hematoxylin and eosin staining and quantified with image analysis software. Immunohistochemical localization of microglia and macrophages was performed using Iba1 staining, and the expression of inflammatory markers was measured after 24 hours of reperfusion by quantitative reverse transcription-polymerase chain reaction. Results— Myeloid-specific MR knockout resulted in a 65% reduction in infarct volume (P=0.005) after middle cerebral artery occlusion. This was accompanied by a significant reduction in activated microglia and macrophages in the ischemic core. Furthermore, myeloid-specific MR knockout suppressed classically activated M1 macrophage markers tumor necrosis factor-&agr;, interleukin-1&bgr;, monocyte chemoattractant protein-1, macrophage inflammatory protein-1&agr;, and interleukin-6 at the same time as partially preserving the induction of alternatively activated, M2, markers Arg1, and Ym1. Conclusions— These data demonstrate that myeloid MR activation exacerbates stroke and identify myeloid MR as a critical target for MR antagonists. Furthermore, these data indicate that MR activation has an important role in controlling immune cell function during the inflammatory response to stroke.

Should the STAIR Criteria Be Modified for Preconditioning Studies

Diverse preconditioning (PC) stimuli protect against a wide variety of neuronal insults in animal models, engendering enthusiasm that PC could be used to protect the brain clinically. Candidate clinical applications include cardiac and vascular surgery, after subarachnoid hemorrhage, and prior to conditions in which acute neuronal injury is anticipated. However, disappointments in clinical validation of multiple neuroprotectants suggest potential problems translating animal data into successful human therapies. Thus, despite strong promise of preclinical PC studies, caution should be maintained in translating these findings into clinical applications. The Stroke Therapy Academic Industry Roundtable working group and the National institute of Neurological Diseases and Stroke proposed working guidelines to improve the utility of preclinical studies that form the foundation of therapies for neurological disease. Here, we review the applicability of these consensus criteria to preconditioning studies and discuss additional considerations for PC studies. We propose that special attention should be paid to several areas, including (1) safety and dosage of PC treatments, (2) meticulously matching preclinical modeling to the human condition to be tested, and (3) timing of both the initiation and discontinuation of the PC stimulus relative to injury ictus.

Von Willebrand Factor Inhibits Mature Smooth Muscle Gene Expression through Impairment of Notch Signaling

Von Willebrand factor (vWF), a hemostatic protein normally synthesized and stored by endothelial cells and platelets, has been localized beyond the endothelium in vascular disease states. Previous studies have implicated potential non-hemostatic functions of vWF, but signaling mechanisms underlying its effects are currently undefined. We present evidence that vWF breaches the endothelium and is expressed in a transmural distribution pattern in cerebral small vessel disease (SVD). To determine the potential molecular consequences of vWF permeation into the vessel wall, we also tested whether vWF impairs Notch regulation of key smooth muscle marker genes. In a co-culture system using Notch ligand expressing cells to stimulate Notch in A7R5 cells, vWF strongly inhibited both the Notch pathway and the activation of mature smooth muscle gene promoters. Similar repressive effects were observed in primary human cerebral vascular smooth muscle cells. Expression of the intracellular domain of NOTCH3 allowed cells to bypass the inhibitory effects of vWF. Moreover, vWF forms molecular complexes with all four mammalian Notch ectodomains, suggesting a novel function of vWF as an extracellular inhibitor of Notch signaling. In sum, these studies demonstrate vWF in the vessel wall as a common feature of cerebral SVD; furthermore, we provide a plausible mechanism by which non-hemostatic vWF may play a novel role in the promotion of vascular disease.

Collagen represses canonical Notch signaling and binds to Notch ectodomain

The Notch signaling system features a growing number of modulators that include extracellular proteins that bind to the Notch ectodomain. Collagens are a complex, heterogeneous family of secreted proteins that serve both structural and signaling functions, most prominently through binding to integrins and DDR. The shared widespread tissue distribution of Notch and collagen prompted us to investigate the effects of collagen on Notch signaling. In a cell co-culture signaling assay, we found that type IV collagen inhibited Notch signaling in H460 and A7R5 cell lines. Moreover, Notch-stimulated expression of mature smooth muscle genes SMA, MHC, SM22, and calponin, which define the physiologic phenotype of normal vascular smooth muscle, was inhibited by type IV collagen in A7R5 cells. Cloned promoters of three of these genes were also inhibited by exposure to collagen. Collagen-dependent repression of Notch signaling required an RBP-jK site within the SM22 promoter. Moreover, repression by collagen required extracellular stimulation of the Notch signaling pathway. Type IV collagen bound to both Notch3 and Jagged1 proteins in purified protein binding assays. In addition, type I collagen also inhibited Notch signaling and bound to Notch and Jagged. We conclude that type IV and type I collagen repress canonical Notch signaling to alter expression of Notch target genes.

Smooth muscle protein 22 alpha-Cre is expressed in myeloid cells in mice.

BACKGROUNDnExperiments using Cre recombinase to study smooth muscle specific functions rely on strict specificity of Cre transgene expression. Therefore, accurate determination of Cre activity is critical to the interpretation of experiments using smooth muscle specific Cre.nnnMETHODS AND RESULTSnTwo lines of smooth muscle protein 22 α-Cre (SM22α-Cre) mice were bred to floxed mice in order to define Cre transgene expression. Southern blotting demonstrated that SM22α-Cre was expressed not only in tissues abundant of smooth muscle, but also in spleen, which consists largely of immune cells including myeloid and lymphoid cells. PCR detected SM22α-Cre expression in peripheral blood and peritoneal macrophages. Analysis of SM22α-Cre mice crossed with a recombination detector GFP mouse revealed GFP expression, and hence recombination, in circulating neutrophils and monocytes by flow cytometry.nnnCONCLUSIONSnSM22α-Cre mediates recombination not only in smooth muscle cells, but also in myeloid cells including neutrophils, monocytes, and macrophages. Given the known contributions of myeloid cells to cardiovascular phenotypes, caution should be taken when interpreting data using SM22α-Cre mice to investigate smooth muscle specific functions. Strategies such as bone marrow transplantation may be necessary when SM22α-Cre is used to differentiate the contribution of smooth muscle cells versus myeloid cells to observed phenotypes.

Hemoglobin Expression in Neurons and Glia After Intracerebral Hemorrhage

The purpose of this study was to examine the expression of hemoglobin (Hb) in the brain after intracerebral hemorrhage (ICH) and the effects of hemin and iron on neuronal Hb. For the in vivo studies, male Sprague-Dawley rats received either a sham operation or an ICH. The rats were killed 1, 4, 24 or 72 h later, and brains were used for real-time polymerase chain reaction (PCR) and immunohistochemistry. For the in vitro study, primary cultured neurons were exposed to either hemin or vehicle. Some neurons also received treatment with deferoxamine, an iron chelator. Neurons were collected 24 h later for real-time PCR. We found that α-globin (HbA) and β-globin (HbB) mRNA levels in the ipsilateral basal ganglia are significantly increased after ICH, and Hb is localized in neurons and glia cells. Exposure of neurons to hemin also upregulated HbA and HbB mRNA levels. Hemin-induced HbA and HbB expression in cultured neurons was reduced by deferoxamine treatment. These results indicate that ICH increases HbA and HbB expression in neurons and glia cells, and that heme and iron may be important factors in inducing endogenous Hb expression after ICH.

Advanced Intimal Hyperplasia Without Luminal Narrowing of Leptomeningeal Arteries in CADASIL

Background and Purpose— Leptomeningeal artery abnormalities in Cerebral Autosomal–Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) have not been extensively characterized. We quantified substructure and diameter of leptomeningeal arteries in CADASIL compared with age-matched controls and the very old; in addition, we characterized intimal thickening in CADASIL using immunohistochemistry. Methods— Frontal and temporal cortex of 6 genetically proven CADASIL brains (average age, 66 years), 6 controls without symptoms of cerebrovascular disease, and 6 very old brains (average age, 89 years) were examined for leptomeningeal artery intimal, medial, and adventitial thickness; inner diameter; and sclerotic index and for smooth muscle markers. Results— The intima of CADASIL arteries was thickened 5-fold compared with controls and the very aged (P<0.0001). Medial thickness was lower in CADASIL compared with controls and the very old (P<0.01). The adventitia was not significantly increased in CADASIL compared with age-matched controls. Arterial diameters were not smaller in CADASIL compared with controls. Sclerotic index was significantly increased in CADASIL compared with other groups (P<0.00001). Intimal cells in CADASIL expressed smooth muscle actin, S100A4, and vimentin but not desmin. Conclusions— Principle changes of leptomeningeal arteries in CADASIL include intimal thickening and medial thinning, but not luminal narrowing. Smooth muscle–like cells participate in neointimal thickening of CADASIL arteries.

The small leucine-rich proteoglycan BGN accumulates in CADASIL and binds to NOTCH3.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited form of cerebral small vessel disease caused by mutations in conserved residues of NOTCH3. Affected arteries of CADASIL feature fibrosis and accumulation of NOTCH3. A variety of collagen subtypes (types I, III, IV, and VI) have been identified in fibrotic CADASIL vessels. Biglycan (BGN) and decorin (DCN) are class I members of the small leucine-rich proteoglycan (SLRP) family that regulate collagen fibril size. Because DCN has been shown to deposit in arteries in cerebral small vessel disease, we tested whether BGN accumulates in arteries of CADASIL brains. BGN was strongly expressed in both small penetrating and leptomeningeal arteries of CADASIL brain. BGN protein was localized to all three layers of arteries (intima, media, and adventitia). Substantially, more immunoreactivity was observed in CADASIL brains compared to controls. Immunoblotting of brain lysates showed a fourfold increase in CADASIL brains (compared to controls). Messenger RNA encoding BGN was also increased in CADASIL and was localized by in situ hybridization to all three vascular layers in CADASIL. Human cerebrovascular smooth muscle cells exposed to purified NOTCH3 ectodomain upregulated BGN, DCN, and COL4A1 through mechanisms that are sensitive to rapamycin, a potent mTOR inhibitor. In addition, BGN protein interacted directly with NOTCH3 protein in cell culture and in direct protein interaction assays. In conclusion, BGN is a CADASIL-enriched protein that potentially accumulates in vessels by mTOR-mediated transcriptional activation and/or post-translational accumulation via protein interactions with NOTCH3 and collagen.