Elaine Hillas

Contribution of Insulin and Akt1 Signaling to Endothelial Nitric Oxide Synthase in the Regulation of Endothelial Function and Blood Pressure

Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues (TTr-IR−/−) or mice with genetic ablation of Akt1 (Akt1−/−). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulin-mediated eNOS phosphorylation in TTr-IR−/− mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1−/− mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulin-stimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid–mediated impairment of eNOS phosphorylation.

Effects of Dietary Sodium and Genetic Background on Angiotensinogen and Renin in Mouse

Elements of a renin-angiotensin system expressed along the entire nephron, including angiotensinogen secreted by proximal tubule and renin expressed in connecting tubule, may participate in the regulation of sodium reabsorption at multiple sites of the nephron. The response of this tubular renin-angiotensin system to stepwise changes in dietary sodium was investigated in 2 mouse strains, the sodium-sensitive inbred C57BL/6 and the sodium-resistant CD1 outbred. Plasma angiotensinogen was not affected by sodium regimen, whereas plasma renin increased 2-fold under low sodium. In both strains, the variation in urinary parameters did not parallel the changes observed in plasma. Angiotensinogen and renin excretion were significantly higher under high sodium than under low sodium. Water deprivation, by contrast, induced significant activation in the tubular expression of angiotensinogen and renin. C57BL/6 exhibited significantly higher urinary excretion of angiotensinogen than did CD1 animals under both conditions of sodium intake. The extent to which these urinary parameters reflect systemic or tubular responses to challenges of sodium homeostasis may depend on the relative contribution of sodium restriction and volume depletion.

Overexpression of mouse angiotensinogen in renal proximal tubule causes salt-sensitive hypertension in mice.

BACKGROUND The role of proximal tubule (PT) angiotensinogen (AGT) in modulating blood pressure has previously been examined using mice expressing PT human AGT and human renin, or rat AGT. These animals are hypertensive; however, the question remains whether alterations in mouse PT AGT alone affects arterial pressure. METHODS Mouse AGT cDNA was knocked-in to the endogenous kidney androgen protein (KAP) gene using an internal ribosomal entry site (IRES)-based strategy. RESULTS The KAP-mAGT animals showed kidney-specific KAP-AGT mRNA expression; renal in situ hybridization detected KAP-AGT mRNA only in PT. Urinary AGT was markedly increased in KAP-mAGT mice. On a high Na diet, radiotelemetric arterial pressure showed a systolic pressure elevation; no significant difference in arterial pressure was observed on a normal diet. Plasma renin concentration (PRC) was reduced in KAP-mAGT animals given a high Na diet, but was not different between mouse lines during normal Na intake. Plasma AGT concentration was not altered by overexpression of PT mouse AGT. CONCLUSIONS In summary, PT overexpression of mouse AGT leads to salt-sensitive hypertension without recruitment of the systemic renin-angiotensin system.

Contribution of Sp1 to initiation of transcription of angiotensinogen

AbstractSeveral genetic polymorphisms have been identified in the proximal promoter of angiotensinogen (AGT). Gene titration experiments in transgenic animals have demonstrated that small increases in the basal expression of AGT can lead to elevated blood pressure. The direct proof that promoter variants of AGT can lead to elevated blood pressure will ultimately require the development of specific animal models. Before such work can be contemplated, however, a formal understanding of the mechanisms controlling transcriptional activation of AGT needs to be developed. Analysis of DNA-protein interactions in vitro and transactivation experiments in cultured cells reveal the critical role of an Sp1 binding site immediately upstream of the TATA box of AGT in both mouse and human. Both sites are required for transcription initiation in the mouse. By contrast, a minimal human AGT promoter can initiate transcription in the absence of either this Sp1 site or the TATA box, albeit at a lower level. Further analysis and consideration of these interspecific differences will be essential for the development of meaningful animal models to probe the mechanism by which AGT may predispose to human essential hypertension.

Response to genetic manipulations of liver angiotensinogen in the physiological range

AbstractGenetic variation in the human angiotensinogen gene (AGT) influences plasma AGT concentration and susceptibility to essential hypertension by a mechanism that remains to be clarified. When one or two additional copies of the gene were inserted by gene titration (by homologous recombination with gap-repair at the AGT locus), both plasma AGT and arterial pressure were elevated in the physiological range in the mouse. The causal dependency between plasma AGT and blood pressure and the relative contribution of the various tissues that express AGT to these two phenotypic parameters remained to be determined. To address these issues, we generated a transgenic mouse with overexpression of the mouse AGT gene restricted to the liver. The transgene was examined in two contrasted genetic backgrounds, the sodium-sensitive C57BL/6J and the sodium-resistant A/J. Transgenic and control male animals underwent continuous cardiovascular monitoring by telemetry for 14 days while under a standard sodium diet (0.2%). Moderate but significant increases in plasma AGT (40%, p = 0.01) and systolic blood pressure (4-6 mmHg, p ranging from 0.01 to <0.001) were observed in the sodium-sensitive background, but not in the sodium-resistant animals. Statistical analysis of a large number of consecutive, repeated measurements of blood pressure afforded power to detect small effects in the physiological range by use of advanced mixed models of analysis of variances and covariances. Although plasma renin activity was increased in the sodium-sensitive background, it did not reach statistical significance. These observations underline a potential contribution of systemic AGT to the mechanism of AGT-mediated hypertension, but the significance of sodium sensitivity in the genetic background suggests participation of the kidney in expression of the elevated blood pressure phenotype, a matter that will warrant further studies. They also highlight the challenge of identifying the contribution of individual genes in complex inheritance, as their effects are modulated by other genetic and environmental determinants.

Natural killer cells attenuate cytomegalovirus-induced hearing loss in mice

Congenital cytomegalovirus (CMV) infection is the most common non-hereditary cause of sensorineural hearing loss (SNHL) yet the mechanisms of hearing loss remain obscure. Natural Killer (NK) cells play a critical role in regulating murine CMV infection via NK cell recognition of the Ly49H cell surface receptor of the viral-encoded m157 ligand expressed at the infected cell surface. This Ly49H NK receptor/m157 ligand interaction has been found to mediate host resistance to CMV in the spleen, and lung, but is much less effective in the liver, so it is not known if this interaction is important in the context of SNHL. Using a murine model for CMV-induced labyrinthitis, we have demonstrated that the Ly49H/m157 interaction mediates host resistance in the temporal bone. BALB/c mice, which lack functional Ly49H, inoculated with mCMV at post-natal day 3 developed profound hearing loss and significant outer hair cell loss by 28 days of life. In contrast, C57BL/6 mice, competent for the Ly49H/m157 interaction, had minimal hearing loss and attenuated outer hair cell loss with the same mCMV dose. Administration of Ly49H blocking antibody or inoculation with a mCMV viral strain deleted for the m157 gene rendered the previously resistant C57BL/6 mouse strain susceptible to hearing loss to a similar extent as the BALB/c mouse strain indicating a direct role of the Ly49H/m157 interaction in mCMV-dependent hearing loss. Additionally, NK cell recruitment to sites of infection was evident in the temporal bone of inoculated susceptible mouse strains. These results demonstrate participation of NK cells in protection from CMV-induced labyrinthitis and SNHL in mice.

A real-time, telemetric method for continuous measurement of portal pressures.

The ability to longitudinally monitor portal and splanchnic pressures would greatly enhance the understanding of acute and chronic liver disease by helping to assess the immediate and long-term impact of therapeutic manipulations. However, a technique for measuring portal pressures in the ambulatory setting is not currently available. To overcome this difficulty, we utilized an approach that involved the implantation of a miniature telemetric device, equipped with a specially-designed pressure transmission catheter, into the spleen of an anesthetized mouse. Using this approach, portal pressures were measured continuously over 5 d in conscious, unrestrained animals, the availability of which will help facilitate studies of the portal circulation requiring long-term stability.