Runx1 is a transcription factor essential for definitive hematopoiesis, and genetic abnormalities in Runx1 cause leukemia. Runx1 is functionally promiscuous and acts as either an oncogene or tumor suppressor gene in certain epithelial cancers. Recent evidence suggests that Runx1 is an important factor in breast cancer, however its role remains ambiguous. Here, we addressed whether Runx1 has a specific pathological role during breast cancer progression and show that Runx1 has an oncogenic function. We observed elevated Runx1 expression in a subset of human breast cancers. Furthermore, throughout the course of disease progression in a classical mouse model of breast cancer (i.e., the MMTV-PyMT transgenic model), Runx1 expression increases in the primary site (mammary gland) and is further upregulated in tumors and distal lung metastatic lesions. Ex vivo studies using tumor epithelial cells derived from these mice express significantly higher levels of Runx1 than normal mammary epithelial cells. The tumor cells exhibit increased rates of migration and invasion, indicative of an aggressive cancer phenotype. Inhibition of Runx1 expression using RNA interference significantly abrogates these cancer-relevant phenotypic characteristics. Importantly, our data establish that Runx1 contributes to murine mammary tumor development and malignancy and potentially represents a key disease-promoting and prognostic factor in human breast cancer progression and metastasis.

Background and Purpose We investigated vasoactive properties of leptomeningeal arterioles (LMAs) under normotensive conditions and during hypertension and aging that are known to have poor collateral flow and little salvageable tissue. Methods LMAs, identified as distal anastomotic arterioles connecting middle and anterior cerebral arteries, were studied isolated and pressurized from young (18 weeks) or aged (48 weeks) normotensive Wistar Kyoto (WKY18, n=14; WKY48, n=6) and spontaneously hypertensive (SHR18, n=16; SHR48, n=6) rats. Myogenic tone and vasoactive responses to pressure as well as endothelial function and ion channel activity were measured. Results LMAs from WKY18 had little myogenic tone at 40 mm Hg (8±3%) that increased in aged WKY48 (30±6%). However, LMAs from both WKY groups dilated to increased pressure and demonstrated little myogenic reactivity, a response that would be conducive to collateral flow. In contrast, LMAs from both SHR18 and SHR48 displayed considerable myogenic tone (56±8% and 43±7%; p<0.01 vs. WKY) and constricted to increased pressure. LMAs from both WKY and SHR groups had similar basal endothelial NO and IK channel activity that opposed tone. However, dilation to sodium nitroprusside, diltiazem and 15 mmol/L KCl was impaired in LMAs from SHR18. Conclusion This study shows for the first time that LMAs from young and aged SHR are vasoconstricted and have impaired vasodilatory responses that may contribute to greater perfusion deficit and little penumbral tissue. These results also suggest that therapeutic opening of pial collaterals is possible during MCAO to create penumbral tissue and prevent infarct expansion.

Severe pre/eclampsia are associated with brain edema that forms preferentially in the posterior cerebral cortex possibly due to decreased sympathetic innervation of posterior cerebral arteries and less effective autoregulation during acute hypertension. In the present study, we examined the effect of pregnancy on the effectiveness of cerebral blood flow autoregulation using laser Doppler flowmetry and edema formation by wet:dry weight in acute hypertension induced by phenylephrine infusion in the anterior and posterior cerebrum from nonpregnant (n=8) and late-pregnant (n=6) Sprague Dawley rats. In addition, we compared the effect of pregnancy on sympathetic innervation by tyrosine hydroxylase staining of posterior and middle cerebral arteries (n=5–6/group) and endothelial and neuronal nitric oxide synthase expression using quantitative polymerase chain reaction (n=3/group). In nonpregnant animals, there was no difference in autoregulation between anterior and posterior cerebrum. However, in late-pregnant animals, the threshold of cerebral blood flow autoregulation was shifted to lower pressures in the posterior cerebrum, which was associated with increased neuronal nitric oxide synthase expression in the posterior cerebral cortex vs. anterior. Compared to the nonpregnant state, pregnancy increased the threshold of autoregulation in both brain regions that was related to decreased expression of endothelial nitric oxide synthase. Lastly, acute hypertension during pregnancy caused greater edema formation in both brain cortices that was not due to changes in sympathetic innervation. These findings suggest that although pregnancy shifted the cerebral blood flow autoregulatory curve to higher pressures in both the anterior and posterior cortices, it did not protect from edema during acute hypertension.

Background and Purpose- Aging and hypertension, comorbidities prevalent in the stroke population, are associated with poor collateral status and worsened stroke outcome. However, underlying mechanisms by which these conditions affect stroke outcome are not clear. We studied the role of PAI (plasminogen activator inhibitor)-1 that is increased in aging and hypertension on brain and vascular expression of inflammatory factors and perfusion that may contribute to worse stroke outcomes. Methods- Aged (≈50 weeks) and young (≈18 weeks) spontaneously hypertensive rats (SHR) were subjected to ischemia by middle cerebral artery occlusion (2 hours) and reperfusion (2 hours) with or without treatment with the PAI-1 inhibitor TM5441. Changes in middle cerebral artery and collateral perfusion territories were measured by multisite laser Doppler. Reactivity to TM5441 was studied using isolated and pressurized leptomeningeal anastomotic arterioles. Brain injury was determined by 2,3,5-triphenyltetrazolium staining and quantitative immunohistochemistry of amyloid-β-42, PAI-1, and hemoglobin. Circulating inflammatory factors were measured by ELISA. Results- Changes in cerebral blood flow during middle cerebral artery occlusion were similar between groups, with both having poor collateral perfusion and incomplete reperfusion. However, aged SHR had greater brain injury versus young (41±2 versus 23±2%, P<0.05) as well as increased brain deposition of amyloid-β-42 and circulating oxLDL (oxidized low-density lipoprotein). Erythrocyte aggregation and hemorrhage within the injured brain was observed in 50% of aged but no young SHR, with increased circulating PAI-1 in this subgroup of aged SHR (16±3 versus 6±2 ng/mL, P<0.05). PAI-1 inhibition with TM5441 improved brain injury but did not affect hemorrhage. TM5441 increased collateral perfusion by 38±7% and dilated leptomeningeal anastomotic arterioles by 44±10%, which was abolished by nitric oxide synthase inhibition. Conclusions- Increased injury in aged SHR seemed to be related to poor collateral perfusion, hemorrhagic transformation, increased amyloid-β-42, and oxidative stress. PAI-1 inhibition reduced infarction in both groups of SHR that possibly due, in part, to increased collateral perfusion.

Alpha smooth muscle actin (alpha-SMA) was recently shown to be present in mouse subcutaneous tissue fibroblasts in the absence of tissue injury. In this study, we used a combination of immunohistochemistry and correlative confocal scanning laser and electron microscopy to investigate the structural organization of alpha-SMA in relation to the nucleus. Furthermore, we explored colocalization analysis as a method for quantifying the amount of alpha-SMA in close approximation to the nucleic acid marker, 4',6-diamidino-2-phenyl-indole, dihydrochloride. Our findings indicate the presence of alpha-SMA within nuclear invaginations in close proximity to the nuclear membrane, but not in the nucleoplasm. Although the function of these alpha-SMA-rich nuclear invaginations is at present unknown, the morphology of these structures suggests their possible involvement in cellular and nuclear mechanotransduction as well as nuclear transport.

Background and Purpose-Previous studies have shown that peroxisome proliferator-activated receptor ␥ (PPAR␥), a ligand-activated transcription factor expressed in vascular cells, is protective of the vasculature. We hypothesized that activation of PPAR␥ could prevent hypertensive remodeling of cerebral arteries and improve vascular function. Methods-Ten female Sprague-Dawley rats were treated with the nitric oxide synthase inhibitor N

Abstract-Previous studies have shown that pregnancy prevents hypertensive remodeling of cerebral arteries. In the present study, we sought to determine whether pregnancy could reverse preexisting remodeling. Nonpregnant virgin Sprague-Dawley rats were treated with the NO synthase inhibitor nitro-L-arginine (0.5 g/L in drinking water) for 2 weeks before mating, after which treatment continued until late gestation for a total of 5 weeks. Pregnant animals with preexisting hypertension (nϭ6) were compared with nonpregnant animals that were treated with nitro-L-arginine for either 2 (nϭ8) or 5 (nϭ9) weeks and compared with nontreated controls (nϭ8). Blood pressure, passive and active diameters, wall thickness, media thickness, and passive distensibility of cerebral arteries were compared between groups. Treatment with nitro-L-arginine caused a significant increase in mean arterial pressure in all of the groups compared with controls that was sustained for the entire study: 103Ϯ3 versus 137Ϯ2, 141Ϯ4, and 140Ϯ7 mm Hg (PϽ0.01). Both 2 and 5 weeks of hypertension caused inward eutrophic remodeling in nonpregnant animals, characterized by decreased inner and outer lumen diameters and no change in media thickness. Pregnancy reversed this remodeling, because late-pregnant animals with preexisting hypertension had inner and outer diameters similar to controls. Passive distensibility was significantly less, and active myogenic tone increased in all of the hypertensive animals, independent of pregnancy. These results demonstrate that pregnancy reverses preexisting hypertensive remodeling of cerebral arteries without a decrease in blood pressure. This reversal of protective remodeling during hypertension in pregnancy may be detrimental by lowering the upper limit of autoregulation, whereas blood pressure remains elevated. Key Words: hypertension pregnancy Ⅲ cerebral arteries Ⅲ remodeling C hronic hypertension is associated with significant remodeling and/or medial hypertrophy of cerebral arteries, which are thought to have a protective function. 1,2 Both remodeling and hypertrophy normalize wall stress that is elevated because of increased blood pressure and shift the autoregulatory curve to the higher range of pressures. 1-4 These processes are also protective of the microcirculation by attenuating increases in downstream pressure. For example, spontaneously hypertensive and stroke-prone spontaneously hypertensive rats, known to undergo medial hypertrophy, were less susceptible to bloodbrain barrier (BBB) disruption during acute hypertension than normotensive Wistar Kyoto rats that did not undergo medial hypertrophy. [5][6][7] In addition, medial hypertrophy is thought to stiffen the vascular wall, making cerebral arteries more resistant to forced dilatation during acute hypertension, thereby protecting the BBB from disruption. 1,2 Hypertension during pregnancy is one of the most common complications of pregnancy, occurring in Յ8% of all pregnancies. 8 It is a unique form of hypertension, somewhere between acute and chronic. I...

Insights into the three-dimensional (3D) organization and function of intracellular structures at nanometer resolution, holds the key to our understanding of the molecular underpinnings of cellular structure-function. Besides this fundamental understanding of the cell at the molecular level, such insights hold great promise in identifying the disease processes by their altered molecular profiles, and help determine precise therapeutic treatments. To achieve this objective, previous studies have employed electron microscopy (EM) tomography with reasonable success. However, a major hurdle in the use of EM tomography is the tedious procedures involved in fixing, high-pressure freezing, staining, serial sectioning, imaging, and finally compiling the EM images to obtain a 3D profile of sub-cellular structures. In contrast, the resolution limit of EM tomography is several nanometers, as compared to just a single or even sub-nanometer using the atomic force microscope (AFM). Although AFM has been hugely successful in 3D imaging studies at nanometer resolution and in real time involving isolated live cellular and isolated organelles, it has had limited success in similar studies involving 3D imaging at nm resolution of intracellular structure-function in situ. In the current study, using both AFM and EM on aldehyde-fixed and semi-dry mouse pancreatic acinar cells, new insights on a number of intracellular structure-function relationships and interactions were achieved. Golgi complexes, some exhibiting vesicles in the process of budding were observed, and small vesicles were caught in the act of fusing with larger vesicles, possibly representing either secretory vesicle biogenesis or vesicle refilling following discharge, or both. These results demonstrate the power and scope of the combined engagement of EM and AFM imaging of fixed semi-dry cells, capable of providing a wealth of new information on cellular structure-function and interactions.