Nicole Bishop

PPARγ Activation Prevents Hypertensive Remodeling of Cerebral Arteries and Improves Vascular Function in Female Rats

Background and Purpose— Previous studies have shown that peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;), a ligand-activated transcription factor expressed in vascular cells, is protective of the vasculature. We hypothesized that activation of PPAR&ggr; 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 NG-nitro-l-arginine methyl ester (l-NAME) for 5 weeks, 8 were treated with l-NAME plus the PPAR&ggr; activator rosiglitazone, and 8 received no treatment and served as controls. Blood pressure, myogenic activity, passive diameters and wall thickness of cerebral arteries, and brain capillary density were compared between the groups. Results— Treatment with l-NAME caused an increase in arterial blood pressure that was sustained with rosiglitazone treatment. l-NAME also caused inward hypertrophic remodeling and enhanced myogenic reactivity of cerebral arteries that was reversed by rosiglitazone. In addition, l-NAME hypertension caused rarefaction of brain capillaries by approximately 12%, whereas treatment with rosiglitazone increased capillary density by approximately 20%. Conclusion— PPAR&ggr; activation may be an effective and clinically relevant way to prevent hypertensive remodeling of cerebral arteries and capillary rarefaction as well as improving vascular function without affecting blood pressure.

Runx1 is associated with breast cancer progression in MMTV‐PyMT transgenic mice and its depletion in vitro inhibits migration and invasion

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. J. Cell. Physiol. 230: 2522–2532, 2015.

Pregnancy Reverses Hypertensive Remodeling of Cerebral Arteries

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 (n=6) were compared with nonpregnant animals that were treated with nitro-l-arginine for either 2 (n=8) or 5 (n=9) weeks and compared with nontreated controls (n=8). 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: 103±3 versus 137±2, 141±4, and 140±7 mm Hg (P<0.01). 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.

Alpha smooth muscle actin distribution in cytoplasm and nuclear invaginations of connective tissue fibroblasts

Alpha smooth muscle actin (α-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 α-SMA in relation to the nucleus. Furthermore, we explored colocalization analysis as a method for quantifying the amount of α-SMA in close approximation to the nucleic acid marker, 4′,6-diamidino-2-phenyl-indole, dihydrochloride. Our findings indicate the presence of α-SMA within nuclear invaginations in close proximity to the nuclear membrane, but not in the nucleoplasm. Although the function of these α-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.

Effect of Pregnancy on Autoregulation of Cerebral Blood Flow in Anterior Versus Posterior Cerebrum

Severe preeclampsia and eclampsia are associated with brain edema that forms preferentially in the posterior cerebral cortex possibly because of 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 per group) and endothelial and neuronal NO synthase expression using quantitative PCR (n=3 per group). In nonpregnant animals, there was no difference in autoregulation between the 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 NO synthase expression in the posterior cerebral cortex versus anterior. Compared with the nonpregnant state, pregnancy increased the threshold of autoregulation in both brain regions that was related to decreased expression of endothelial NO synthase. Lastly, acute hypertension during pregnancy caused greater edema formation in both brain cortices that was not attributed 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.

Pial Collateral Reactivity During Hypertension and Aging: Understanding the Function of Collaterals for Stroke Therapy

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) rats and spontaneously hypertensive rats (SHR18, n=16; SHR48, n=6). 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 versus WKY) and constricted to increased pressure. LMAs from both WKY and SHR groups had similar basal endothelial nitric oxide and IK channel activity that opposed tone. However, dilation to sodium nitroprusside, diltiazem and 15 mmol/L KCl was impaired in LMAs from SHR18. Conclusions— 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 middle cerebral artery occlusion to create penumbral tissue and prevent infarct expansion.

3D organization and function of the cell: Golgi budding and vesicle biogenesis to docking at the porosome complex

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.

The influence of pregnancy and gender on perivascular innervation of rat posterior cerebral arteries

The authors investigated the influence of pregnancy and gender on the density of trigeminal and sympathetic perivascular nerves in posterior cerebral arteries (PCA) and the reactivity to norepinephrine and calcitonin gene-related peptide (CGRP). PCAs were isolated from nonpregnant, late-pregnant, postpartum, and male rats, mounted and pressurized on an arteriograph chamber to obtain concentration-response curves to norepinephrine and CGRP. Arteries were immunostained for CGRP-, tyrosine hydroxylase—, and protein gene product 9.5 (PGP 9.5)—containing perivascular nerves, and nerve density was determined morphologically. Pregnancy had a trophic effect on trigeminal perivascular innervation (P < .01 vs male); however, this was not accompanied by a change in reactivity to CGRP. Sympathetic and PGP 9.5 nerve densities were not altered by pregnancy or gender, and there were no differences in reactivity to norepinephrine. Together, these results suggest that the increase in trigeminal innervation during pregnancy is more related to nociception than in controlling resting cerebral blood flow.

Aquaporin-assisted and ER-mediated mitochondrial fission: A hypothesis

It is well established that the status of the endoplasmic reticulum (ER) and mitochondria, and the interactions between them, is critical to numerous cellular functions including apoptosis. Mitochondrial dynamics is greatly influenced by cell stress, and recent studies implicate ER in mitochondrial fission. Although a number of proteins have been identified to participate in ER-induced mitochondrial fission, the molecular mechanism of the process is little understood. In the current study, we confirm the involvement of ER in mitochondrial fission and hypothesize the involvement of water channels or aquaporins (AQP) in the process. Previous studies demonstrate the presence of AQP both in the ER and mitochondrial membranes. Mitochondrial swelling has been observed following mitochondrial calcium overload, and studies report that chelation of cytosolic calcium induces extensive mitochondrial division at ER contact sites. Based on this information, the involvement of ER in mitochondrial division, possibly via water channels, is hypothesized. Utilizing a multi-faceted imaging approach consisting of atomic force microscopy on aldehyde-fixed and semi-dry cells, transmission electron microscopy, and immunofluorescence microscopy on live cells, the physical interactions between the two organelles are demonstrated. Mitochondrial fission following ER stress was abrogated with exposure of cells to the AQP inhibitor mercuric chloride, suggesting the involvement of AQP(s) especially AQP8 and AQP9 known to be present in the mitochondrial membrane, in mitochondrial fission.

Sex-dependent, zinc-induced dephosphorylation of phospholamban by tissue-nonspecific alkaline phosphatase in the cardiac sarcomere.

We have previously reported that Zn(2+) infused into the coronary arteries of isolated rat hearts leads to the potent dephosphorylation of phospholamban (PLB) as well as a noticeable but less potent dephosphorylation of the ryanodine receptor 2. We hypothesized in the present study that a Zn(2+)-activated phosphatase is located in the vicinity of the sarcoplasmic reticulum (SR) where PLB and ryanodine receptor 2 reside. We report here the novel finding of tissue-nonspecific alkaline phosphatase (TNAP), a zinc-dependent enzyme, localized to the SR in the cardiac sarcomere of mouse myocardium. TNAP activity was enhanced by injection of Zn acetate into a tail vein before harvesting the heart and imaged using electron microscopy of electron dense deposits indicative of the hydrolysis of exogenous β-glycerophosphate. TNAP activity was observed localized to the ends of the Z-line corresponding to SR and was qualitatively more visible in myocardium of males compared with females. Correspondingly, PLB phosphorylation status was potently reduced in myocardium of males injected with Zn acetate, whereas there was no apparent effect of Zn acetate injection on PLB phosphorylation in females. Surprisingly, Western blot analysis of TNAP content suggested a significantly lower TNAP content in males compared with females. These data suggest that TNAP plays a role in governing the phosphorylation status of calcium handling proteins in the SR. Furthermore, the content and activity of TNAP are differentially regulated between the sexes and thus may account for some sex differences in cardiopathologies associated with calcium handling.