Transgenic mice expressing an intracellular fluorescent fusion of angiotensin II demonstrate renal thrombotic microangiopathy and elevated blood pressure.

We have generated transgenic mice that express angiotensin II (ANG II) fused downstream of enhanced cyan fluorescent protein, expression of which is regulated by the mouse metallothionein promoter. The fusion protein, which lacks a secretory signal, is retained intracellularly. In the present study, RT-PCR, immunoblot analyses, whole-animal fluorescent imaging, and fluorescent microscopy of murine embryonic fibroblasts confirm expression of the fusion protein in vivo and in vitro. The transgene is expressed in all tissues tested (including brain, heart, kidney, liver, lung, and testes), and radioimmunoassay of plasma samples obtained from transgenic mice indicate no increase in circulating ANG II over wild-type levels, consistent with intracellular retention of the transgene product. Kidneys from transgenic and corresponding wild-type littermates were histologically evaluated, and abnormalities in transgenic mice consistent with thrombotic microangiopathy were observed; microthrombosis was frequently observed within the glomerular capillaries and small vessels. In addition, systolic and diastolic blood pressures, measured by telemetry (n = 8 for each group), were significantly higher in transgenic mice compared with wild-type littermates. Blood pressure of line A male transgenic mice was 125 + or - 1.7 over 97 + or - 1.6 compared with 109 + or - 1.7 over 83 + or - 1.4 mmHg in wild-type littermates (systolic over diastolic). In summary, overexpression of an intracellular fluorescent fusion protein of ANG II correlates with elevated blood pressure and kidney pathology. This transgenic model may be useful to further explore the intracellular renin-angiotensin system and its implication in abnormal kidney function and hypertension.

In vitro evidence for an intracellular site of angiotensin action.

To differentiate the relative effects of nuclear and cell surface angiotensin II (Ang II) receptors, we mutated the angiotensinogen cDNA by removing the signal sequence-encoding region to produce a nonsecreted form of angiotensinogen [Ang(-S)Exp]. Rat hepatoma cells (which produce renin and angiotensin-converting enzyme mRNAs) were stably transfected with Ang(-S)Exp/pSVL (or a corresponding control) expression plasmid, and mitotic indices were measured for stably transfected cell lines. Experimental clonal cell lines demonstrate an average of 33+/-4.4% (P<0.001) increase in percentage-labeled nuclei compared with control cell lines. The mitogenic effect is blocked by 10(-6) mol/L losartan and by 1 micromol/L renin antisense phosphorothioate oligomers but not by 10(-6) mol/L candesartan. In addition, phenylarsine oxide, which blocks angiotensin receptor internalization, abolishes the losartan inhibitory effect, suggesting that after cell-surface receptor-mediated endocytosis, losartan blocks Ang II nuclear receptors. PDGF mRNA levels are elevated 2.2-fold in Ang(-S)Exp transfected cell lines; addition of anti-PDGF antibodies to the culture medium partially blocks the mitogenic effect of Ang(-S)Exp, while anti-Ang II antibodies have no effect. These results suggest that the Ang(-S)Exp growth effect is due, in part, to autocrine/paracrine stimulation by secreted PDGF after Ang II/Ang II receptor intracellular interactions. We further demonstrate that these cells produce the alternative renin transcript, renin 1A, which apparently lacks a signal sequence and is maintained intracellularly. Collectively, these studies of cultured cells suggest that some cell types may possess components of the renin-angiotensin system that permit intracellular processing of angiotensinogen to Ang II and that Ang II generated intracellularly may be mitogenic.

The clinical implication of tissue renin angiotensin systems.

The presence, and in many cases the regulated synthesis, of components of the renin-angiotensin system have been demonstrated in multiple tissues, indicating the existence of tissue angiotensin-generating systems. These vary with respect to which renin-angiotensin system components are synthesized locally and which are taken up from plasma. Enzymes unrelated to the classical renin-angiotensin system may also contribute to tissue angiotensin synthesis. However, based on the available data, the prevailing opinion that kidney-derived renin is in all cases the only physiologically relevant renin in tissues must be revised. Also there is evidence indicating a role for tissue angiotensin systems in the pathogenesis of cardiovascular disease and in cardiovascular structural remodeling. The angiotensin-regulated synthesis of aldosterone in cardiac tissue has been described, suggesting the possibility that a renin-angiotensin-aldosterone system exists in the heart. In addition, intracellular (intracrine) sites of angiotensin action have been reported. Some of these findings have implications for therapeutics and, in particular, for the use of angiotensin converting-enzyme inhibitors and angiotensin receptor blockers. Finally, tissue angiotensin systems outside the cardiovascular system also appear to be physiologically relevant.

The effect of education on patients' willingness to participate in a telemedicine study.

We assessed the effect of previous education on patients' willingness to participate in a clinical study of telemedicine for hypertensive patients. The design was a cross-sectional study of adult patients arriving for appointments in a hypertension clinic. Of the 259 patients approached, 86% completed a baseline survey and were subsequently asked if they would be willing to participate in a one-year telemedicine study. One hundred and fifty patients (58%) agreed to participate. A stepwise logistic regression analysis was performed to assess the effect of level of education on willingness to participate, while controlling for potentially confounding variables. In the final model, only education remained significant. The odds ratio for non-participation of patients with high-school education or less was 3.6 (95% confidence interval 1.9-7.0). Educational status should be carefully considered when designing, implementing and interpreting telemedicine studies.

Testing efficacy with detection controlled estimation: an application to telemedicine.

Detection controlled estimation (DCE) is a powerful new econometric estimator in the family of missing data estimators. By collecting measures from a variety of inspectors or inspection technologies, DCE is able to make inferences about the entire population, even when that population is not directly observed. Using this innovative method, we were able to assess whether telemedicine technology could be substituted for in-person visits when providing maintenance care for patients with hypertension. Our findings indicate that there is no support for the proposition that telemedicine is less effective than in-person visits for determining whether patients have high blood pressure. Indeed, our results imply that telemedicine misses 7% fewer cases of high blood pressure than in-person visits do. The results of this study indicate that DCE may be an effective tool for use in cost-effectiveness or cost-benefit analysis in health care.

Patient and physician satisfaction in a clinical study of telemedicine in a hypertensive patient population.

We studied patient and physician satisfaction with telemedicine for the care of a hypertensive population. Once recruited, participants were seen both in person and via telemedicine (in random order) on the same day. After each meeting, patient and physician satisfaction surveys were completed. In the 12-month study, there were 107 pairs of visits. The physicians reported a small but significant increase in workload, mental effort, technical skills and visit duration for telemedicine when compared with face-to-face consultations. They noted that the telemedicine system worked well in the majority of cases and could reduce the need for future treatment. Patients reported slightly but significantly higher satisfaction scores for the following for in-person than for telemedicine meetings: technical quality, interpersonal care and time spent. Patients reported high satisfaction scores for both telemedicine and in-person visits.

A method to report utilization for quality initiatives in medical facilities.

OBJECTIVE: We undertook this project to outline a methodology for quantifying aggregate health care utilization of medical "technologies" that could be rank ordered by volume. The identification of specific high-volume technologies could guide future efforts for quality initiatives such as program planning, preventive services implementation, quality improvement activities, and innovative and cost-effective technology development. DESIGN: This study utilized a retrospective cross-sectional study design. METHODS: We generated combined ranks for the top 200 high-volume procedures from three data sources that incorporated in- and outpatient procedures. Data were collected using primarily ICD-9 and CPT-4 codes; all codes were translated into CPT-4 codes and collapsed into categories using truncated three-digit CPT-4 codes. Frequencies for each collapsed code were determined with each dataset; procedures were reranked based on the mean rank of the three sources. MAIN OUTCOME MEASURES: We itemized the individual procedure codes making up each of the top 20 categories and reported the unique codes making up at least 80% of the procedure code category. RESULTS: The top five procedure categories identified in this study were patient visits (inpatient and outpatient), chest x-rays, mammograms, ophthalmological services, and electrocardiograms. CONCLUSION: The methodology described provides a new way to combine and concisely report on utilization of procedures that is relevant to data obtained from different sources. This methodology may be of potential benefit to health care administrators, technology developers, and other planners as they contemplate ways to identify quality and technology development initiatives that can have a broad impact on populations served by health care organizations.

Effects of chromosomal integration site upon p53 interactions with DNA consensus sequence homologies.

In the present study, we report that, despite the presence of one perfect p53 consensus sequence homology (designated SCL CS) and four half-sites within the 3'-untranslated region of the stem cell leukemia (SCL) gene, the native endogenous gene is not regulated by p53. We employ a tet-repressible system to show that, under conditions in which the WAF1 mRNA steady-state level is upregulated fourfold by p53, the SCL mRNA level is not altered. In a previous report, we demonstrated that p53 interactions with the SCL CS can upregulate downstream reporter gene activity 43-fold in transient reporter assays. This disparity prompted us to explore the differences between p53 regulation of SCL CS activity in organized (chromosomally integrated) and disorganized (non-replicating episomal plasmid) chromatin. We show that p53 can increase (between 3-80-fold), decrease (between 5-33-fold) or have no effect upon transactivation of an SCL CS/reporter fusion gene depending upon chromosomal integration site. Most studies used to characterize p53 binding sites employ transient transfection assays. Our results suggest that characterization of consensus sequence homologies by assay of transiently transfected cells may be inaccurate.

Renin-angiotensin system: genes to bedside.

Atherosclerosis and its vascular sequela are responsible for considerable morbidity and mortality rates. Several risk factors have been implicated in the pathogenesis of atherosclerosis, and the search for other risk factors continues on the medical horizon. Renin-angiotensin system (RAS), a multienzyme, multilocale axis, has been extensively studied as an important mediator of atherosclerosis. Recently, the tissue-based angiotensin system has been suggested as the most significant pathway of RAS. A genetic polymorphism in the human gene for the angiotensin-converting enzyme (ACE), one of the two enzymes of RAS, has been found to have a strong association with higher risk for acute coronary events, sudden cardiac death, vascular restenosis after angioplasty, and idiopathic and hypertrophic cardiomyopathy. Clinical and animal data support angiotensin II to be the final common pathway in the enzyme cascade of RAS and ACE as the key enzyme in the generation of Angiotensin II. ACE gene polymorphism appears to modify expression of cellular and free ACE levels and could represent a genetic marker for cardiovascular disease.

The application of molecular genetic techniques to the study of hypertensive diseases.

The techniques of modern molecular genetics are shedding new light on hypertension and its sequelae. This article discusses techniques which have identified genes associated with hypertension and have pointed the way toward identifying the full cohort of genes operative in all forms of human hypertension. These techniques have expanded understanding of the pathophysiology of hypertension as well as its prevention.

Suppression of cellular proliferation using p53 DNA recognition site-related oligonucleotides.

A number of oligonucleotides were designed to bind through Hoogsteen triple helix or Watson-Crick hydrogen bonds to the p53 consensus sequence homology localized within the human nontranscribed rRNA spacer region. The oligomers, which bind in vitro to the consensus sequence homology, function as p53 analogues in cells deficient in wild-type p53 protein. Oligomers suppress proliferation of human colon cancer cells by three to eightfold, but only suppress proliferation of normal human mesangial cells or lung fibroblasts by less than 50%. On the basis of these studies, p53 analogues may be used therapeutically to selectively modify proliferation of transformed cells.