Use of a biological peptide pump to study chronic peptide hormone action in transgenic mice. Direct and indirect effects of angiotensin II on the heart.

Angiotensin II is a peptide hormone regulator of blood pressure and fluid balance in mammals. Evidence obtained largely in vitro has also suggested that angiotensin II has growth-promoting effects and that it might thereby contribute to such pathological phenomena as cardiac hypertrophy, a major risk factor for cardiovascular mortality. It has been difficult to test for the direct growth-promoting effects of angiotensin II in vivo, however, because of the generalized effects of the peptide on hemodynamics. To overcome this limitation and to test for cardiac-specific functions of angiotensin II, we generated transgenic mice expressing an angiotensin II-producing fusion protein exclusively in cardiac myocytes. Our findings are the first to distinguish between local and systemic effects of angiotensin II on the heart and introduce a novel technique for studying tissue-specific peptide function.

Development and application of a biological peptide pump for the study of the in vivo actions of angiotensin peptides.

In the past few years, a great deal of interest has been focused on the possibility that angiotensin peptides could have direct effects on target tissues independent of their hemodynamic effects. In addition, there has been much speculation on the potential biological roles of angiotensin peptides other than angiotensin II. Unfortunately, a direct test of these possibilities in whole animals has been difficult due to limitations in existing biological systems. In this review, we describe the characteristics of an engineered protein capable of directing the production of a wide variety of peptides to specific tissues and cell types in transgenic animals and discuss its potential applications.

Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies.

M. Zhang, D. Methot, V. Poppa, Y. Fujio, K. Walsh and C. E. Murry. Cardiomyocyte Grafting for Cardiac Repair: Graft Cell Death and Anti-Death Strategies. Journal of Molecular and Cellular Cardiology (2001) 33, 907-921. Recent studies indicate that cardiomyocyte grafting forms new myocardium in injured hearts. It is unknown, however, whether physiologically significant amounts of new myocardium can be generated. Pilot experiments showed that death of grafted rat neonatal cardiomyocytes limited formation of new myocardium after acute cryoinjury. Time-course studies showed that, at 30 min after grafting, only 1.8(+/-0.4)% of graft cells were TUNEL-positive. At 1 day, however, TUNEL indices increased to 32.1(+/-3.5)% and remained high at 4 days, averaging 9.8(+/-3.8)%. By 7 days, TUNEL decreased to 1.0(+/-0.2)%. Electron microscopy revealed that dead cells had features of both irreversible ischemic injury and apoptosis. To test whether ischemia contributed to poor graft survival, grafts were placed into vascularized 2-week-old cardiac granulation tissue or normal myocardium. TUNEL indices were reduced by 53% and 86%, respectively. Adenoviral infection of graft cells with the cytoprotective kinase Akt, or constitutively active Akt, reduced TUNEL indices by 31% and 40%, respectively, compared to beta -gal-transfected controls. Neither treatment reached statistical significance compared to untreated controls, however. Heat shock reduced cardiomyocyte death in vitro in response to serum deprivation, glucose depletion, and viral activation of the Fas death pathway. When cardiomyocytes were heat shocked prior to grafting, graft cell death in vivo was reduced by 54% at day 1. Therefore, high levels of cardiomyocyte death occur for at least 4 days after grafting into injured hearts, in large part due to ischemia. Death can be limited by activating the Akt pathway and even more effectively by heat shock prior to transplantation.

Knockout of renin-angiotensin system genes: effects on vascular development.

Pharmacologic inhibition of the renin-angiotensin system (RAS) is a widely accepted and effective treatment for hypertension. However, in the past several years, much attention has been focused on additional roles of the RAS including the possibility that its end-product, angiotensin II, could elicit end-organ pathologies independent of its effect on blood pressure. The ability to selectively delete genes in mice (by homologous recombination or gene knockouts) has led to new--and sometimes surprising--insights into the roles of the RAS in the developmental modeling and pathologic remodeling of the heart and blood vessels.

In vivo enzymatic assay reveals catalytic activity of the human renin precursor in tissues.

The aspartyl protease renin is secreted into the circulation of mammals in 2 forms: the proteolytically processed active form of the enzyme and the precursor form, prorenin. Prorenin has no detectable enzymatic activity in the circulation, but it is the exclusive form of the enzyme produced by several tissues that also produce the other components of the renin enzymatic cascade (renin-angiotensin system). To test whether prorenin might be enzymatically active in these tissues, transgenic mice expressing the human renin substrate (angiotensinogen) exclusively in the pituitary gland were mated to mice expressing either active human renin or prorenin in the same tissue. Measurement of in vivo product formation in pituitary glands of double-transgenic mice revealed that human prorenin was enzymatically active, and Western blot analysis demonstrated that this prorenin was in the precursor form with its prosegment attached. This in vivo enzymatic assay demonstrates for the first time that human prorenin can be activated within tissues by nonproteolytic means, where it could contribute to the activity of a localized renin-angiotensin system.

Local Delivery of TGF-b Antibodies to Prevent Neointima Formation after Balloon Injury in a Pig Coronary Artery Model.

BACKGROUND: The formation of neointima after vessel injury results from smooth muscle cell proliferation and extracellular matrix secretion. This process is activated by multiple growth factor release. Among these, Transforming Growth Factor-b (TGF-b) has been shown to play an important role. We hypothesized that local delivery of TGF-b antibodies could reduce neointima formation after balloon angioplasty. METHODS AND RESULTS: Using autoperfusion double-balloon catheters (Baxter, Irvine, California), we infused polyclonal TGF-b antibodies in 30 minutes, immediately after oversized balloon angioplasty in pig coronary arteries. Eleven coronary arteries received 100 m anti-TGF-b and thirteen served as controls. Animals were sacrificed 10 weeks later; coronary segments were harvested and processed for histologic quantitative assessment of the neointima. The extent of injury was similar in treated versus control vessels (39% +/- 5% vs. 30% +/- 4%) and there was no difference in intimal thickening (0.63 +/- 0.19 mm for treated vs. 0.52 +/- 0.12 mm for controls). A previously validated restenosis injury index (ratio of neointimal area to total wall area over extent of injury) was also similar in both groups, 1.46 +/- 0.15 for treated versus 1.55 +/- 0.14 for controls. CONCLUSION: Local delivery of a single dose of TGF-b antibodies failed to demonstrate a benefit on neointima formation in a pig coronary artery model.

Tissue targeting of angiotensin peptides.

Angiotensin II (Ang II) is an octapeptide generated by the sequential proteolytic action of renin and angiotensin converting enzyme on the glycoprotein angiotensinogen. While numerous mammalian tissues have been shown to express some or all of the components of the renin-angiotensin system (RAS), the function of most of these tissue RAS remains a matter of conjecture. To test for tissue-specific functions of Ang II and as an alternative to co-expressing all the components of RAS, we have engineered a fusion protein that leads to direct Ang II release within specific tissues. The angiotensin peptide is cleaved from the fusion protein within the secretory pathway by the ubiquitous endoprotease furin and is released from the cell by constitutive secretion. Direct injection of an expression vector encoding such a fusion protein into rat cardiac ventricles results in a highly localized expression of atrial natriuretic peptide mRNA (an angiotensin responsive marker of cardiac hypertrophy), demonstrating the utility of this approach for local targeting of mature peptides to tissues in animal models.

Evaluation of tyrosinase minigene co-injection as a marker for genetic manipulations in transgenic mice.

The utility of tyrosinase minigene co-injection was evaluated as a visual marker for the generation and breeding of transgenic mice. In an evaluation of 39 transgenic founder animals and 44 transgenic lines five phenotypic patterns of pigmentation were consistently observed, including albino, dark, light, mottled and himalayan. In these studies co-injection of the tyrosinase minigene along with the transgene of interest (TOI) resulted in genomic integration of the two transgenes in 95% of the F0 generation. Co-segregation of transgenes occurred in 94% of doubly transgenic mice in the F1 generation, without dissociation in subsequent generations. All pigmented phenotypes proved useful for distinguishing homozygous from heterozygous F2 animals via backcross trials, while the light, mottled and himalayan phenotypes proved useful in visually discriminating between homozygous and heterozygous F2 animals. In addition, the light, mottled and himalayan phenotypes proved useful in determining segregation patterns of transgenes in the progeny of crosses between separate transgenic lines. Moreover, there appears to be a correlation between intensity of pigmentation and degree of expression of the co-injected TOI. These studies confirm that tyrosinase co-injection is a useful adjunct in transgenic mouse studies and can serve to reduce routine genetic validation of transgenic lines.