Alissa Kapke

Endothelial Nitric Oxide Synthase Regulates Brain-Derived Neurotrophic Factor Expression and Neurogenesis after Stroke in Mice

Here, we investigate the effects of endothelial nitric oxide synthase (eNOS) on angiogenesis, neurogenesis, neurotrophic factor expression, and neurological functional outcome after stroke. Wild-type and eNOS knock-out (eNOS-/-) mice were subjected to permanent occlusion of the right middle cerebral artery. eNOS-/- mice exhibited more severe neurological functional deficit after stroke than wild-type mice. Decreased subventricular zone (SVZ) progenitor cell proliferation and migration, measured using bromodeoxyuridine, Ki-67, nestin, and doublecortin immunostaining in the ischemic brain, and decreased angiogenesis, as demonstrated by reduced endothelial cell proliferation, vessel perimeter, and vascular density in the ischemic border, were evident in eNOS-/- mice compared with wild-type mice. eNOS-deficient mice also exhibited a reduced response to vascular endothelial growth factor (VEGF)-induced angiogenesis in a corneal assay. ELISAs showed that eNOS-/- mice have decreased brain-derived neurotrophic factor (BDNF) expression but not VEGF and basic fibroblast growth factor in the ischemic brain compared with wild-type mice. In addition, cultured SVZ neurosphere formation, proliferation, telomerase activity, and neurite outgrowth but not cell viability from eNOS-/- mice were significantly reduced compared with wild-type mice. BDNF treatment of SVZ cells derived from eNOS-/- mice restored the decreased neurosphere formation, proliferation, neurite outgrowth, and telomerase activity in cultured eNOS-/- SVZ neurospheres. SVZ explant cell migration also was significantly decreased in eNOS-/- mice compared with wild-type mice. These data indicate that eNOS is not only a downstream mediator for VEGF and angiogenesis but also regulates BDNF expression in the ischemic brain and influences progenitor cell proliferation, neuronal migration, and neurite outgrowth and affects functional recovery after stroke.

One-Year Follow-Up After Bone Marrow Stromal Cell Treatment in Middle-Aged Female Rats With Stroke

Background and Purpose— We sought to evaluate the long-term effects of bone marrow stromal cell (BMSC) treatment on retired breeder rats with stroke. Methods— Female retired breeder rats were subjected to 2-hour middle cerebral artery occlusion (MCAO) followed by an injection of 2×106 male BMSCs (n=8) or phosphate-buffered saline (n=11) into the ipsilateral internal carotid artery at 1 day after stroke. The rats were humanely killed 1 year later. Functional tests, in situ hybridization, and histochemical and immunohistochemical staining were performed. Results— Significant recovery of neurological deficits was found in BMSC-treated rats beginning 2 weeks after cell injection compared with control animals. The beneficial effects of cell transplantation persisted for at least 1 year (P<0.01). In situ hybridization for the Y chromosome showed that donor cells survived in the brains of recipient rats, among which 22.3±1.95% of cells expressed the astrocyte marker glial fibrillary acidic protein, 16.8±2.13% expressed the neuronal marker microtubule-associated protein 2, and 5.5±0.42% and <1% of cells colocalized with the microglial marker IB4 and the endothelial cell marker von Willebrand factor, respectively. Only very few BMSCs, however, were found in peripheral organs such as the heart, lung, liver, spleen, and kidney in recipient rats. BMSCs significantly reduced axonal loss (P<0.01), the thickness of the lesion scar wall (P<0.01), and the number of Nogo-A–positive cells (P<0.05) along the scar border; meanwhile, synaptophysin expression (P<0.05) was significantly increased in BMSC-treated ischemic brains compared with control untreated brains. Conclusions— The beneficial effects of BMSCs on ischemic brain tissue persisted for at least 1 year. Most surviving BMSCs were present in the ischemic brain, but very few were found in other organs. The long-term improvement in functional outcome may be related to the structural and molecular changes induced by BMSCs.

Antifibrotic Effects of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline on the Heart and Kidney in Aldosterone-Salt Hypertensive Rats

Abstract-N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits not only hematopoietic cell proliferation but also fibroblast proliferation and collagen synthesis in vitro. Ac-SDKP also prevents collagen deposition and cell proliferation in the left ventricle (LV) in rats with renovascular hypertension (renin dependent). However, it is not clear whether Ac-SDKP has similar effects in a model of renin-independent hypertension (aldosterone-salt). Using a hypertensive rat model of cardiac and renal fibrosis created by chronic elevation of circulating aldosterone (ALDO) levels, we examined the effect of Ac-SDKP on blood pressure, cardiac and renal fibrosis and hypertrophy, and proliferating cell nuclear antigen (PCNA) expression in the LV and left kidney. Uninephrectomized rats were divided into 4 groups: (1) controls that received tap water, (2) rats that received ALDO (0.75 &mgr;g/h SC) and 1% NaCl/0.2% KCl in drinking water (ALDO-salt), (3) rats that received ALDO-salt plus Ac-SDKP 400 &mgr;g · kg−1 · day−1 SC, and (4) rats that received ALDO-salt plus Ac-SDKP 800 &mgr;g · kg−1 · d−1 SC. After 6 weeks of treatment, the ALDO-salt group was found to have significantly increased blood pressure with decreased body weight and plasma renin concentration (P <0.05), LV and renal hypertrophy as well as renal injury, significantly increased collagen content in both ventricles and kidney as well as increased collagen volume fraction in the LV (P <0.0001), and significantly increased interstitial and perivascular PCNA-positive cells in the LV and kidney (P <0.0001). Ac-SDKP at 800 &mgr;g · kg−1 · d−1 markedly prevented cardiac and renal fibrosis (P <0.005) without affecting blood pressure or organ hypertrophy. It also suppressed PCNA expression in the LV and kidney in a dose-dependent manner. We concluded that Ac-SDKP prevents increased collagen deposition and cell proliferation in the heart and kidney in ALDO-salt hypertensive rats. Because ACE inhibitors increase plasma and tissue Ac-SDKP and decrease cardiac and renal fibrosis, we speculate that Ac-SDKP may participate in the antifibrotic effect of ACE inhibitors.

Role of AT2 Receptors in the Cardioprotective Effect of AT1 Antagonists in Mice

Angiotensin II (Ang II) acts mainly on two receptor subtypes: AT1 and AT2. Most of the known biological actions of Ang II are mediated by AT1 receptors; however, the role of AT2 receptors remains unclear. We tested the hypothesis that the cardioprotective effects of AT1 receptor antagonists (AT1-ant) after myocardial infarction (MI) are partially mediated by activation of AT2 receptors; thus in AT2 receptor gene knockout mice (AT2−/Y), the effect of AT1-ant will be diminished or absent. MI was induced by ligating the left anterior descending coronary artery. Four weeks later, AT2−/Y and their wild-type littermates (AT2+/Y) were started on vehicle, AT1-ant (valsartan, 50 mg/kg per day), or ACE inhibitor (enalapril, 20 mg/kg per day) for 20 weeks. Basal blood pressure and cardiac function as well as remodeling after MI did not differ between AT2+/Y and AT2−/Y. AT1-ant increased ejection fraction and cardiac output and decreased left ventricular diastolic dimension, myocyte cross-sectional area, and interstitial collagen deposition in AT2+/Y, and these effects were significantly diminished in AT2−/Y. ACE inhibitors improved cardiac function and remodeling similarly in both strains. We concluded that (1) activation of AT2 during AT1 blockade plays an important role in the therapeutic effect of AT1-ant and (2) the AT2 receptor may not play an important role in regulation of cardiac function, either under basal conditions after MI remodeling or in the therapeutic effect of ACE inhibitors.

Bone marrow stromal cells upregulate expression of bone morphogenetic proteins 2 and 4, gap junction protein connexin-43 and synaptophysin after stroke in rats

Bone morphogenetic proteins play a key role in astrocytic differentiation. Astrocytes express the gap junctional protein connexin-43, which permits exchange of small molecules in brain and enhances synaptic efficacy. Bone marrow stromal cells produce soluble factors including bone morphogenetic protein 2 and bone morphogenetic protein 4 (bone morphogenetic protein 2/4) in ischemic brain. Here, we tested whether intra-carotid infusion of bone marrow stromal cells promotes synaptophysin expression and neurological functional recovery after stroke in rats. Adult male Wistar rats were subjected to 2 h of right middle cerebral artery occlusion. Rats were treated with or without bone marrow stromal cells at 24 h after middle cerebral artery occlusion via intra-arterial injection (n=8/group). A battery of functional tests was performed. Immunostaining of 5-bromo-2-deoxyuridine, Ki67, bone morphogenetic protein 2/4, connexin-43, synaptophysin, glial fibrillary acidic protein, neuronal nuclear antigen, and double staining of 5-bromo-2-deoxyuridine/glial fibrillary acidic protein, 5-bromo-2-deoxyuridine/neuronal nuclear antigen, glial fibrillary acidic protein/bone morphogenetic protein 2/4 and glial fibrillary acidic protein/connexin-43 were employed. Rats treated with bone marrow stromal cells significantly (P<0.05) improved functional recovery compared with the controls. 5-Bromo-2-deoxyuridine and Ki67 positive cells in the ipsilateral subventricular zone were significantly (P<0.05) increased in bone marrow stromal cell treatment group compared with the controls, respectively. Administration of bone marrow stromal cells significantly (P<0.05) promoted the proliferating cell astrocytic differentiation, and increased bone morphogenetic protein 2/4, connexin-43 and synaptophysin expression in the ischemic boundary zone compared with the controls, respectively. Bone morphogenetic protein 2/4 expression correlated with the expression of connexin-43 (r=0.84, P<0.05) and connexin-43 expression correlated with the expression of synaptophysin (r=0.73, P<0.05) in the ischemic boundary zone, respectively. Administration of bone marrow stromal cells via an intra-carotid route increases endogenous brain bone morphogenetic protein 2/4 and connexin-43 expression in astrocytes and promotes synaptophysin expression, which may benefit functional recovery after stroke in rats.

Nitric Oxide Donor Upregulation of Stromal Cell-Derived Factor-1/Chemokine (CXC Motif) Receptor 4 Enhances Bone Marrow Stromal Cell Migration into Ischemic Brain After Stroke

Stromal cell‐derived factor‐1 (SDF1) and its chemokine (CXC motif) receptor 4 (CXCR4), along with matrix metalloproteinases (MMPs), regulate bone marrow stromal cell (BMSC) migration. We tested the hypothesis that a nitric oxide donor, DETA‐NONOate, increases endogenous ischemic brain SDF1 and BMSC CXCR4 and MMP9 expression, which promotes BMSC migration into ischemic brain and thereby enhances functional outcome after stroke. C57BL/6J mice were subjected to middle cerebral artery occlusion (MCAo), and 24 hours later, the following were intravenously administered (n = 9 mice per group): (a) phosphate‐buffered saline; (b) BMSCs (5 × 105); (c) 0.4 mg/kg DETA‐NONOate; (d) combination of CXCR4‐inhibition BMSCs with DETA‐NONOate; and (e) combination of BMSCs with DETA‐NONOate. To elucidate the mechanisms underlying combination‐enhanced BMSC migration, transwell cocultures of BMSC with mouse brain endothelial cells (MBECs) or astrocytes were performed. Combination treatment significantly improved functional outcome after stroke compared with BMSC monotherapy and MCAo control, and it increased SDF1 expression in the ischemic brain compared with DETA‐NONOate monotherapy and MCAo control. The number of BMSCs in the ischemic brain was significantly increased after combination BMSC with DETA‐NONOate treatment compared with monotherapy with BMSCs. The number of engrafted BMSCs was significantly correlated with functional outcome after stroke. DETA‐NONOate significantly increased BMSC CXCR4 and MMP9 expression and promoted BMSC adhesion and migration to MBECs and astrocytes compared with nontreatment BMSCs. Inhibition of CXCR4 or MMPs in BMSCs significantly decreased DETA‐NONOate‐induced BMSC adhesion and migration. Our data demonstrate that DETA‐NONOate enhanced the therapeutic potency of BMSCs, possibly via upregulation of SDF1/CXCR4 and MMP pathways, and increased BMSC engraftment into the ischemic brain.

MRI identification of white matter reorganization enhanced by erythropoietin treatment in a rat model of focal ischemia

Background and Purpose— The objectives of the present study were to: (1) noninvasively identify white matter reorganization and monitor its progress within 6 weeks after the onset of stroke; and (2) quantitatively investigate the effect of recombinant human erythropoietin treatment on this structural change using in vivo measurement of diffusion anisotropy. Methods— Male Wistar rats were subjected to middle cerebral artery occlusion and treated with recombinant human erythropoietin intraperitoneally at a dose of 5000 U/kg of body weight (n=11) or the same volume of saline (n=7) daily for 7 days starting 24 hours after middle cerebral artery occlusion. MRI measurements of T2- and diffusion-weighted images and cerebral blood flow were performed and neurological severity score was assessed at 1 day and weekly for 6 weeks after middle cerebral artery occlusion. Luxol fast blue and Bielschowsky staining were used to demonstrate myelin and axons, respectively. Results— White matter reorganization occurred along the ischemic lesion boundary after stroke. The region of white matter reorganization seen on the tissue slice coincided with the elevated area on the fractional anisotropy map, which can be accurately identified. The increase in elevated fractional anisotropy pixels corresponded with progress of white matter reorganization and was associated with improvement of neurological function. Treatment with recombinant human erythropoietin after stroke significantly enhanced white matter reorganization, restored local cerebral blood flow, and expedited functional recovery. Conclusions— White matter reorganization can be detected by fractional anisotropy. Elevated fractional anisotropy pixels may be a good MRI index to stage white matter remodeling and predict functional outcome.

Atorvastatin downregulates tissue plasminogen activator-aggravated genes mediating coagulation and vascular permeability in single cerebral endothelial cells captured by laser microdissection.

The effects of statins on gene expression of cerebral endothelial cells (ECs) in vivo have not been investigated after stroke. We developed a rapid double immunofluorescent staining protocol with antibodies against von Willebrand factor (a marker for endothelium) and glial fibrillary acidic protein (a marker for astrocytes) for laser capture microdissection to isolate single ECs in brain tissue of the rat. Using this protocol in combination with real-time PCR, we found that stroke significantly increased mRNA levels of protease-activated receptor 1 (PAR-1) and tissue factor (TF) in ECs isolated from ischemic cerebral microvessels compared with nonischemic vessels. Treatment of embolic stroke with recombinant human tissue plasminogen activator (rht-PA) 4 h after stroke further elevated PAR-1 mRNA levels nearly 1000-fold in the core and 500-fold in the boundary above the nonstroke group 30 h after stroke, while TF mRNA levels were elevated approximately 10 fold above the nonstroke group. Furthermore, stroke significantly increased matrix metalloproteinase (MMP) 2 and 9 mRNA levels in the ischemic core and boundary regions 6 and 30 h after stroke. Treatment with rht-PA-upregulated MMP2 expression in the ischemic boundary and core. Atorvastatin completely blocked rht-PA upregulation of the above genes, when atorvastatin in combination with rht-PA was administered 4 h after stroke. Monotherapy of atorvastatin 4 h after stroke did not significantly reduce expression of genes examined in the present study. These data provide evidence that atorvastatin reduces exogenous tPA-aggravated cerebral endothelial genes that mediate thrombosis and blood-brain barrier permeability, which could contribute to the beneficial effects of statins on thrombolytic treatment of acute stroke.

Breast cancer incidence in a cohort of women with benign breast disease from a multiethnic, primary health care population.

Abstract:  Women with benign breast diseases (BBD), particularly those with lesions classified as proliferative, have previously been reported to be at increased risk for subsequent development of breast cancer (BC). A cohort of 4970 women with biopsy‐proven BBD, identified after histopathology review of BBD biopsies, was studied for determination of subsequent development of BC. We report on 4537 eligible women, 28% of whom are African‐American, whose BBD mass was evaluable for pathologic assessment of breast tissue. Ascertainment of subsequent progression to BC from BBD was accomplished through examination of the tumor registries of the Henry Ford Health system, the Detroit SEER registry, and the State of Michigan cancer registry. Incidence rates (IR) are reported per 100,000 person years at risk (100 k pyr). Poisson regression models were used to evaluate the association of demographic and lesion characteristics with BC incidence, using person years at the time of BBD diagnosis as the offset variable. The estimated overall BC IR for this cohort is 452 (95% confidence interval [CI] = 394–519) per 100 k pyr. Incidence for women age 50 and older is 80% greater than for younger women (p = 0.007, IRR = 1.8, 95% CI = 1.36–2.36). Neither marital status (p = 0.91, IRR = 0.97, 95% CI = 0.73–1.29) nor race (p = 0.67, IRR = 0.9, 95% CI = 0.54–1.48) is associated with differences in BC IR. Compared with women having nonproliferative lesions, the risk for BC is greater for women with atypical ductal hyperplasia of (IRR = 5.0; 95%CI = 2.26–11.0; p < 0.001) and other proliferative lesions (IR = 1.7, 95% CI = 1.02–2.95; p = 0.04). BC risk for woman with atypical lesions is significantly higher than for women with proliferative lesions without atypia (IRR = 2.58, 95% CI = 1.35–4.90; p = 0.0039). Neither race nor marital status was a factor for BC incidence from BBD in this cohort. Age retained its importance as a predictor of risk. BBD lesion histopathology in the outcome categories of either proliferative without atypia or proliferative with atypia are significant risk factors for BC, even when adjusted for the influence of demographic characteristics. The risks associated with BBD histological classifications were not different across races.

Synergistic Effect of an Endothelin Type A Receptor Antagonist, S-0139, With rtPA on the Neuroprotection After Embolic Stroke

Background and Purpose— Using a model of embolic stroke, the present study tested the hypothesis that blockage of endothelin-1 with S-0139, a specific endothelin type A receptor (ETA) antagonist, enhances the neuroprotective effect of recombinant tissue plasminogen activator (rtPA) by suppressing molecules that mediate thrombosis and blood brain barrier (BBB) disruption induced by ischemia and rtPA. Methods— Rats (n=104) subjected to embolic middle cerebral artery (MCA) occlusion were randomly divided into 1 of 4 infusion groups with 26 rats per group: (1) the control group in which rats were administered saline, (2) the monotherapy rtPA group in which rtPA was intravenously administered at a dose of 10 mg/kg 4 hours after MCA occlusion, (3) the monotherapy S-0139 group in which S-0139 was intravenously given 2 hours after MCA occlusion, and (4) the combination of rtPA +S-0139 group in which S-0139 and rtPA were given 2 and 4 hours after MCA occlusion, respectively. Measurements of infarct volume and parenchymal hemorrhage, behavioral outcome, and immunostaining were performed on rats euthanized 1 and 7 days after stroke. Results— The combination therapy of S-0139 and rtPA significantly (P<0.01) reduced infarct volume (24.8±0.9% versus 33.8±1.5% in control) and hemorrhagic area (7.1±6.1 &mgr;m2 versus 36.5±19.2 &mgr;m2 in control) and improved functional recovery compared with control saline-treated animals. Immunostaining analysis revealed that the combination therapy had the synergistically suppressed ischemia- and rtPA-induced ICAM-1, protease-activated receptor 1 (PAR-1), as well as accumulation of platelets in cerebral microvessels. Furthermore, the combination treatment synergistically reduced loss of laminin, ZO1, and occludin in cerebral vessels. Conclusions— These data suggest that S-0139 provides the neuroprotection by suppressing ischemia- and rtPA-triggered molecules that evoke thrombosis and BBB disruption.