Andrew S. Greene

Opposing Actions of Angiotensin II on Microvascular Growth and Arterial Blood Pressure

We performed studies to further elucidate the mechanisms of angiotensin II (Ang II)-induced angiogenesis of the microvasculature. Rats were placed on a high salt diet (4% NaCl), and Ang II was infused at a subpressor rate (5 ng/kg per minute) for 3 days. Blood pressure was measured daily for 2 control and 3 infusion days. Microvessel density in the cremaster muscle was measured at the end of the infusion. Vessel density in rats that received subpressor Ang II infusion increased by 12.6% compared with rats that received vehicle infusion. When the angiotensin type 2 (AT2) receptor antagonist PD 123319 was coinfused with Ang II, blood pressure was elevated and vessel density increased above that observed with Ang II infusion alone (23% increase). When the AT1 receptor antagonist losartan was coinfused with Ang II, blood pressure was lower than control and vessel density was reduced compared with the Ang II group but was still greater than control (7.8% increase). In this study, Ang II stimulated angiogenesis in the rat cremaster muscle; this effect was enhanced by AT2 antagonism and inhibited by AT1 antagonism. Ang II infusion at a subpressor dose resulted in a pressor response with AT2 antagonism and a depressor response with AT1 antagonism. This suggests that in the microvasculature, the AT1 receptor mediates angiogenesis and vasoconstriction, and the AT2 receptor mediates an inhibition of angiogenesis and vasodilation.

Brown Norway Chromosome 13 Confers Protection From High Salt to Consomic Dahl S Rat

Consomic rats (SS.BN13), in which chromosome 13 from normotensive inbred Brown Norway rats from a colony maintained at the Medical College of Wisconsin (BN/Mcw) was introgressed into the background of Dahl salt-sensitive (SS/Mcw) rats, also maintained in a colony at the Medical College of Wisconsin, were bred. The present studies determined the mean arterial pressure (MAP) responses to salt and renal and peripheral vascular responses to norepinephrine and angiotensin II; 24-hour protein excretion and histological analyses were used to assess renal pathology in rats that received a high salt (4% NaCl) diet for 4 weeks. MAP of rats measured daily during the fourth week averaged 170±3.3 mm Hg in SS/Mcw rats, 119±2.1 mm Hg in SS.BN13 rats, and 103±1.3 mm Hg in BN/Mcw rats. After salt depletion, MAP fell an average of 27±4.5 mm Hg in SS/Mcw rats, 9±2.6 mm Hg in SS.BN13 rats, and 11±3.0 mm Hg in BN/Mcw rats. Protein excretion of SS/Mcw rats on a high salt diet averaged 189±30 mg/24 h, 63±18 mg/24 h in SS.BN13 rats, and 40±6.4 mg/24 h in BN/Mcw rats. Compared with SS.BN13 and BN/Mcw rats, SS/Mcw rats exhibited significantly greater increases of renal vascular resistance in response to intravenous norepinephrine and angiotensin II. Severe medullary interstitial fibrosis and tubular necrosis after a high salt diet were found consistently in SS/Mcw rat kidneys but were largely absent in the SS.BN13 and BN/Mcw rat kidneys. A similar degree of glomerular sclerosis was found in both SS/Mcw and SS.BN13 rats. In rats fed a 0.4% salt diet, the glomerular filtration rate of SS/Mcw rats was significantly less than that of BN/Mcw and SS.BN13 rats. These results reveal a powerful gene, or set of genes, within chromosome 13 of BN/Mcw rats that confers protection from the detrimental effects of high salt to the SS/Mcw rats.

MicroRNA–target pairs in the rat kidney identified by microRNA microarray, proteomic, and bioinformatic analysis

Mammalian genomes contain several hundred highly conserved genes encoding microRNAs. In silico analysis has predicted that a typical microRNA may regulate the expression of hundreds of target genes, suggesting miRNAs might have broad biological significance. A major challenge is to obtain experimental evidence for predicted microRNA-target pairs. We reasoned that reciprocal expression of a microRNA and a predicted target within a physiological context would support the presence and relevance of a microRNA-target pair. We used microRNA microarray and proteomic techniques to analyze the cortex and the medulla of rat kidneys. Of the 377 microRNAs analyzed, we identified 6 as enriched in the renal cortex and 11 in the renal medulla. From approximately 2100 detectable protein spots in two-dimensional gels, we identified 58 proteins as more abundant in the renal cortex and 72 in the renal medulla. The differential expression of several microRNAs and proteins was verified by real-time PCR and Western blot analyses, respectively. Several pairs of reciprocally expressed microRNAs and proteins were predicted to be microRNA-target pairs by TargetScan, PicTar, or miRanda. Seven pairs were predicted by two algorithms and two pairs by all three algorithms. The identification of reciprocal expression of microRNAs and their computationally predicted targets in the rat kidney provides a unique molecular basis for further exploring the biological role of microRNA. In addition, this study establishes a differential profile of microRNA expression between the renal cortex and the renal medulla and greatly expands the known differential proteome profiles between the two kidney regions.

Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays

BackgroundLong oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear.ResultsUnmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 μm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR.ConclusionsMicroarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied.

Suppression of Angiotensin-Converting Enzyme Expression and Activity by Shear Stress

Shear stress caused by the frictional forces of a fluid moving over a cell monolayer is an important regulator of gene expression. In this study, we investigated the effect of shear stress on angiotensin-converting enzyme (ACE) expression and promoter activity in vitro and on local vascular ACE activity in vivo. ACE activity measured in bovine pulmonary artery endothelial (BPAE) cells was reduced by 49.5% after exposure to a shear stress of 20 dyne/cm2 for 18 hours. Short-term shearing (2 hours) elevated ACE activity in BPAE cells, whereas long-term shearing produced a time-dependent reduction in ACE activity by 23.3%, 33.5%, and 48.9% at 8, 12, and 18 hours, respectively. Northern blot analysis revealed that shear stress (20 dyne/cm2 for 18 hours) significantly reduced ACE mRNA expression by 82%. To determine the mechanism of ACE activity and message reduction, the effect of shear on transcriptionally related events was determined in a rabbit aortic endothelial cell line (W3LUC) stably transfected with 1.3 kb of a rat ACE promoter/luciferase construct. Different shear stress magnitudes (5 to 20 dyne/cm2) caused suppression of luciferase activity by an average of 40.7%. ACE promoter activity was suppressed by 2 hours of shear stress (24.7%) and was further inhibited at time periods > 8 hours. In vivo elevations in shear stress were created by placing a stainless steel clip over a 12-mm region of the rat abdominal aorta. Restriction of vessel diameter increased blood flow velocity and caused reduction in vascular ACE activity by 40%. These studies suggest that elevations in the level of shear stress alter endothelial cell function by suppressing ACE gene and protein expression in vitro and in vivo.

MR-derived cerebral blood volume maps: Issues regarding histological validation and assessment of tumor angiogenesis

In an effort to develop MRI methods for the evaluation of tumor angiogenesis (new blood vessel formation), MRI‐derived cerebral blood volume (CBV) information has been compared to histologic measures of microvessel density (MVD). Although MVD is a standard marker of angiogenesis, it is not a direct correlate of the volume measurements made with MRI, and therefore inappropriate for the development and validation of the MR techniques. Therefore, the goal of this study was to develop an approach by which MR measurements of CBV can be directly correlated. To this end, dynamic susceptibility contrast (DSC) MRI experiments were performed in six Fisher rats implanted with 9L gliosarcoma brain tumors. Subsequently, the circulation was perfused with a latex compound (Microfil®), after which 50‐μm tissue sections were analyzed for vessel count, diameter, and the fraction of area comprised of vessels. The results demonstrate that while fractional area (FA) does not provide a good measure of CBV, FA corrected for section thickness effects does. Whereas the FA in normal brain was found to be 13.03 ± 1.83% the corrected FA, or fractional volume (FV), was 1.89 ± 0.39%, a value in agreement with those reported in the literature for normal brain. Furthermore, while no significant difference was found between normal brain and tumor FA (P = 0.55), the difference was significant for FV (P = 0.036), as would be expected. And only with FV does a correlation with the MRI‐derived CBV become apparent (rS = 0.74). There was strong correlation (rs = 0.886) between the tumor / normal blood volume ratios as estimated by each technique, although the MR‐ratio (1.56 ± 0.29) underestimated the histologic‐ratio (2.35 ± 0.75). Thus, the correlation of MRI CBV methods requires a measurement of fractional vessel area and correction of this area for section thickness effects. This new independent correlative measure should enable efficient and accurate progress in the development of MRI methods to evaluate tumor angiogenesis. Magn Reson Med 46:735–747, 2001.

Angiogenesis Induced by Electrical Stimulation Is Mediated by Angiotensin II and VEGF

Objective: Physiological angiogenesis in skeletal muscle is an adaptive response to physical training and electrical stimulation. This study investigated the role of angiotensin II (Ang II) in regulating both angiogenesis and vascular endothelial growth factor (VEGF) protein expression induced by electrical stimulation.

Carotid sinus baroreceptor reflex control of respiration.

We have studied the effect of the carotid sinus baroreceptor reflex on respiration in 10 vagotomized, spontaneously breathing, pentobarbital anesthetized dogs. The carotid body chemoreceptor reflex response was eliminated by surgically excluding the carotid bodies from the carotid sinus baroreceptor area. Steady state frequency, tidal volume, and minute ventilation were measured after 25 mm Hg step changes in intrasinus pressure between 50 and 200 mm Hg. Over this range, the step decreases in intrasinus pressure caused concomitant increases in mean arterial pressure from 86 to 182 mm Hg. All of the respiratory response curves were sigmoidal in shape. Decreasing intrasinus pressure from 200 to 50 mm Hg caused respiratory frequency to increase from 4.8 to 9.7/min, and tidal volume to decrease from 704 to 515 ml. The calculated total ventilation, however, increased from 3180 to 4530 ml/min. The time of inspiration decreased from 3.7 to 2.4 seconds, and the time of expiration decreased from 9.8 to 4.1 seconds. These ventilatory responses are shown to be baroreceptor reflex mediated, and not secondary to changes in arterial pressure. These findings indicate that not only does the carotid sinus baroreceptor reflex control arterial pressure, but it also simultaneously influences ventilation, through changes in both respiratory frequency and tidal volume.

HYPOXIA INCREASES THE ACTIVITY OF CA2+-SENSITIVE K+ CHANNELS IN CAT CEREBRAL ARTERIAL MUSCLE CELL MEMBRANES

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O2, PO2, on the predominant K+ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K+-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NPo) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]i) between 0 and 100 μM, but was significantly increased upon elevation of intracellular free Ca2+ concentration ([Ca2+]i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O2 from 21% to < 2% reversibly increased the NPo, mean open time, and event frequency of the Ca2+-sensitive, high-conductance single-channel K+ current recorded at a patch potential of + 20 mV. A similar reduction in PO2 also produced a transient increase in the activity of the 215-pS K+ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca2+]i or intracellular pH (pHi) of cat cerebral arterial muscle cells, as measured using Ca2+- or pH-sensitive fluorescent probes. Reduced PO2 caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO2 increases the activity of a high-conductance, Ca2+-sensitive K+ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca2+]i and pHi.

MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor β1 (TGFβ1), a model of epithelial–mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFβ1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA–target interactions. Knockdown of miR-382, which was up-regulated by TGFβ1, attenuated TGFβ1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3′-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGFβ1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFβ1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFβ1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGFβ1.