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. 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. Angiotensin II (Ang II) 1The abbreviations used are: Ang II, angiotensin II; RAS, renin-angiotensin system; AT, angiotensin receptor; ANP, atrial natriuretic peptide; fs, frog skin; [Ca2+]i, intracellular free calcium concentration. 1The abbreviations used are: Ang II, angiotensin II; RAS, renin-angiotensin system; AT, angiotensin receptor; ANP, atrial natriuretic peptide; fs, frog skin; [Ca2+]i, intracellular free calcium concentration. is the peptide product of the renin-angiotensin system (RAS) and is generated via two sequential proteolytic steps. First, renin, a circulating aspartyl protease, cleaves the decapeptide angiotensin I (Ang I) from the amino terminus of the hepatic glycoprotein angiotensinogen. Angiotensin-converting enzyme then removes two amino acids from the carboxyl terminus of Ang I to release the vasoactive peptide Ang II. This enzymatic cascade that occurs in the circulation has also been suggested to take place within certain tissues. Brain, kidney, adrenal and pituitary glands, heart, vasculature and reproductive tissues have been shown to express protein and/or mRNA for many of the components of the RAS including angiotensin receptors (1Ganong W.F. Deschepper C.F. Steele M.K. Intebi A. Am. J. Hypertens. 1989; 2: 320-322Crossref PubMed Scopus (30) Google Scholar, 2Ganong W.F. Horm. Res. ( Basel ). 1989; 31: 24-31Crossref PubMed Scopus (41) Google Scholar, 3Mulrow P.J. Yale J. Biol. Med. 1989; 62: 503-510PubMed Google Scholar, 4Paul M. Wagner J. Dzau V.J. J. Clin. Invest. 1993; 91: 2058-2064Crossref PubMed Scopus (217) Google Scholar, 5Griendling K.K. Murphy T.J. Alexander R.W. Circulation. 1993; 87: 1816-1828Crossref PubMed Scopus (333) Google Scholar, 6Gomez R.A. Norwood V.F. Am. J. Kidney Dis. 1995; 26: 409-431Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Activity of local or tissue RAS has been implicated in a variety of physiologic pathways and pathophysiologic conditions including sympathetic nerve transmission, pituitary hormone secretion, migration of eggs in the oviduct, renal development, hypertension, end-stage renal disease, cardiac hypertrophy, and restenosis following vascular injury (reviewed in Refs. 7Ganong W.F. Front. Neuroendocrinol. 1993; 14: 233-249Crossref PubMed Scopus (111) Google Scholar, 8Lee M.A. Bohm M. Paul M. Ganten D. Circulation. 1993; 87: IV7-13PubMed Google Scholar, 9Neuringer J.R. Brenner B.M. Am. J. Kidney Dis. 1993; 22: 98-104Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 10Ganong W.F. Neurosci. Biobehav. Rev. 1995; 19: 241-250Crossref PubMed Scopus (53) Google Scholar). However, two findings make it difficult to differentiate between the possibilities that the Ang II mediating these activities is either generated and acts locally, comes from the circulation, or is synthesized locally and acts elsewhere to generate the observed effects. First, in tissues, components of the RAS are often expressed in different cell types or are present in only extremely low levels, making it difficult to be certain that all of the necessary components would encounter each other in biologically relevant concentrations. Second, renin is synthesized as a zymogen that is activated before secretion from the juxtaglomerular cells of the kidney. Removal of the kidneys results in virtual disappearance of renin from the circulation, whereas its precursor, prorenin, remains (11Sealey J.E. Rubattu S. Am. J. Hypertens. 1989; 2: 358-366Crossref PubMed Scopus (65) Google Scholar, 12Campbell D.J. Kladis A. Skinner S.L. Whitworth J.A. J. Hypertens. 1991; 9: 265-274Crossref PubMed Scopus (61) Google Scholar, 13Hsueh W.A. Baxter J.D. Hypertension. 1991; 17: 469-479Crossref PubMed Google Scholar, 14Hosoi M. Kim S. Tabata T. Nishitani H. Nishizawa Y. Morii H. Murakami K. Yamamoto K. J. Clin. Endocrinol. & Metab. 1992; 74: 680-684PubMed Google Scholar). This finding has raised the question about whether non-renal tissues have the capacity to activate prorenin and thereby carry out the first reaction in the RAS. For these reasons and despite much circumstantial evidence, the functions of tissue RAS remain a matter of conjecture.By using transgenic animals, it is possible to test for the biological effect of tissue expression of the RAS by inducing either loss of function or gain of function mutations. Mice lacking all RAS activity have been generated via homologous recombination by insertional mutagenesis of the angiotensinogen gene (the only known substrate for generation of the angiotensin peptides) (15Tanimoto K. Sugiyama F. Goto Y. Ishida J. Takimoto E. Yagami K. Fukamizu A. Murakami K. J. Biol. Chem. 1994; 269: 31334-31337Abstract Full Text PDF PubMed Google Scholar, 16Kim H.S. Krege J.H. Kluckman K.D. Hagaman J.R. Hodgin J.B. Best C.F. Jennette J.C. Coffman T.M. Maeda N. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2735-2739Crossref PubMed Scopus (580) Google Scholar). These mice are hypotensive and exhibit some defects in the development of the kidney, but these experiments shed little light on the normal physiologic functions of tissue RAS. On the other hand, generalized overexpression of components of the RAS in transgenic mice and rats is clearly linked to an increase in blood pressure (17Mullins J.J. Peters J. Ganten D. Nature. 1990; 344: 541-544Crossref PubMed Scopus (807) Google Scholar, 18Ganten D. Wagner J. Zeh K. Bader M. Michel J.B. Paul M. Zimmermann F. Ruf P. Hilgenfeldt U. Ganten U. Kaling M. Bachmann S. Fukamizu A. Mullins J.J. Murakami K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7806-7810Crossref PubMed Scopus (200) Google Scholar, 19Keusch, G., Kurtz, A., Fehr, J., Eckardt, K. U., Frei, D., Bauer, C., and Binswanger, U. (1989) Nephron , 51, Suppl. 1, 29–33.Google Scholar, 20Fukamizu A. Sugimura K. Takimoto E. Sugiyama F. Seo M.-S. Takahashi S. Hatae T. Kajiwara N. Yagami K. Murakami K. J. Biol. Chem. 1993; 268: 11617-11621Abstract Full Text PDF PubMed Google Scholar). To discriminate between circulatory and local effects of transgene expression, it is necessary to target expression of the RAS transgenes to specific tissues. However, this approach is complicated by the fact that all of the RAS components (i.e. angiotensinogen, prorenin, angiotensin-converting enzyme, Ang II receptors, and a prorenin convertase) need to be expressed in the target tissue. In addition, the Ang II generated can elicit biological responses at other sites.As an alternative to co-expressing RAS components, we have engineered a fusion protein that leads to the direct release of an Ang II peptide within specific tissues. This peptide is a natural variant of Ang II found in the skin of the Australian frog Crinia georgiana(fsAngII (21Erspamer V. Melchiorri P. Nakajima T. Yasuhara T. ( Basel ). PubMed Scopus Google has been to have activity to that of mammalian Ang II Yasuhara T. Nakajima T. J. Med. Chem. PubMed Scopus Google Scholar). a for proteolytic release of from the fusion protein by the ubiquitous furin T. T. J. M. Murakami K. K. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). In this the and biological activity of the peptide and experiments to test for the effect of local of in Direct injection of the expression vector into the of rat leads to local expression of and of atrial natriuretic peptide expression, a marker of cardiac in cardiac ventricles P. J. Clin. Invest. PubMed Scopus Google Scholar, Deschepper C.F. Am. J. Google Scholar). These results for the first the of using an engineered fusion protein to a peptide local biological activity in have engineered a fusion protein that leads to the direct release of an Ang II within tissues. The angiotensin peptide used in this is released by a by the to the constitutive secretion of the released a mammalian of the is a expressed in all tissues and cell J.A. J. Clin. Invest. PubMed Scopus Google Scholar, J. P.J. J. Biol. 1990; PubMed Scopus Google Scholar, K. M. T. M. A. Murakami K. K. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). for of by furin have been and the amino from to to the has been shown to to the in cells T. Murakami K. K. 1993; PubMed Scopus Google Scholar, S. K. T. Murakami K. K. J. ( ). 1994; PubMed Scopus Google Scholar). II, is an from the skin of the Australian frog (21Erspamer V. Melchiorri P. Nakajima T. Yasuhara T. ( Basel ). PubMed Scopus Google its is a substrate for by furin T. T. J. M. Murakami K. K. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). results that and secretion of from the fusion protein the of a between the and the angiotensin peptide to the The of the prorenin as a by the engineered furin in this peptide V. Baxter J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the in the fusion protein for a of fusion in cell and by the of protein or for the release of peptide is also by the finding that of into of rat leads to intracellular and secretion of results that to the Ang II While the a to that of mammalian Ang II Ang to the is to most of the effects of Ang II P. J. T. Hypertension. PubMed Scopus Google M. P.J. Best C.F. Maeda N. Smithies O. Coffman T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google occurs Ang II However, only of the release of intracellular its of action on target cells is that of Ang II The of these the in biological of Ang II and on and Yasuhara T. Nakajima T. J. Med. Chem. PubMed Scopus Google have that the activity of the in rats that of Ang in injection using rat cardiac for the first that an engineered fusion protein can be to a mature peptide and have local biological effects. are as to the of the cells the peptide in as for either the or angiotensin peptides to a low of expression and/or the secretion of the fusion protein from the However, as the used in expression vector has a cell it be expressed in either cardiac or of rats in blood to or in circulating Ang II between the and In to the of a increase in mRNA in the of the gene expression to the injection the to an increase in to these that the expression of is highly localized to cells the injection These the in results of J. Y. H.S. S. 1993; Full Text PDF PubMed Scopus Google the effects of Ang II in cardiac in that in local overexpression of in the rat leads to a highly localized cardiac we have a expression vector that can as an alternative to co-expressing all the components of the RAS to generate Ang II. This of approach also be the of tissue-specific in transgenic to test the of other peptides on Angiotensin II (Ang II) 1The abbreviations used are: Ang II, angiotensin II; RAS, renin-angiotensin system; AT, angiotensin receptor; ANP, atrial natriuretic peptide; fs, frog skin; [Ca2+]i, intracellular free calcium concentration. 1The abbreviations used are: Ang II, angiotensin II; RAS, renin-angiotensin system; AT, angiotensin receptor; ANP, atrial natriuretic peptide; fs, frog skin; [Ca2+]i, intracellular free calcium concentration. is the peptide product of the renin-angiotensin system (RAS) and is generated via two sequential proteolytic steps. First, renin, a circulating aspartyl protease, cleaves the decapeptide angiotensin I (Ang I) from the amino terminus of the hepatic glycoprotein angiotensinogen. Angiotensin-converting enzyme then removes two amino acids from the carboxyl terminus of Ang I to release the vasoactive peptide Ang II. This enzymatic cascade that occurs in the circulation has also been suggested to take place within certain tissues. Brain, kidney, adrenal and pituitary glands, heart, vasculature and reproductive tissues have been shown to express protein and/or mRNA for many of the components of the RAS including angiotensin receptors (1Ganong W.F. Deschepper C.F. Steele M.K. Intebi A. Am. J. Hypertens. 1989; 2: 320-322Crossref PubMed Scopus (30) Google Scholar, 2Ganong W.F. Horm. Res. ( Basel ). 1989; 31: 24-31Crossref PubMed Scopus (41) Google Scholar, 3Mulrow P.J. Yale J. Biol. Med. 1989; 62: 503-510PubMed Google Scholar, 4Paul M. Wagner J. Dzau V.J. J. Clin. Invest. 1993; 91: 2058-2064Crossref PubMed Scopus (217) Google Scholar, 5Griendling K.K. Murphy T.J. Alexander R.W. Circulation. 1993; 87: 1816-1828Crossref PubMed Scopus (333) Google Scholar, 6Gomez R.A. Norwood V.F. Am. J. Kidney Dis. 1995; 26: 409-431Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Activity of local or tissue RAS has been implicated in a variety of physiologic pathways and pathophysiologic conditions including sympathetic nerve transmission, pituitary hormone secretion, migration of eggs in the oviduct, renal development, hypertension, end-stage renal disease, cardiac hypertrophy, and restenosis following vascular injury (reviewed in Refs. 7Ganong W.F. Front. Neuroendocrinol. 1993; 14: 233-249Crossref PubMed Scopus (111) Google Scholar, 8Lee M.A. Bohm M. Paul M. Ganten D. Circulation. 1993; 87: IV7-13PubMed Google Scholar, 9Neuringer J.R. Brenner B.M. Am. J. Kidney Dis. 1993; 22: 98-104Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 10Ganong W.F. Neurosci. Biobehav. Rev. 1995; 19: 241-250Crossref PubMed Scopus (53) Google Scholar). However, two findings make it difficult to differentiate between the possibilities that the Ang II mediating these activities is either generated and acts locally, comes from the circulation, or is synthesized locally and acts elsewhere to generate the observed effects. First, in tissues, components of the RAS are often expressed in different cell types or are present in only extremely low levels, making it difficult to be certain that all of the necessary components would encounter each other in biologically relevant concentrations. Second, renin is synthesized as a zymogen that is activated before secretion from the juxtaglomerular cells of the kidney. Removal of the kidneys results in virtual disappearance of renin from the circulation, whereas its precursor, prorenin, remains (11Sealey J.E. Rubattu S. Am. J. Hypertens. 1989; 2: 358-366Crossref PubMed Scopus (65) Google Scholar, 12Campbell D.J. Kladis A. Skinner S.L. Whitworth J.A. J. Hypertens. 1991; 9: 265-274Crossref PubMed Scopus (61) Google Scholar, 13Hsueh W.A. Baxter J.D. Hypertension. 1991; 17: 469-479Crossref PubMed Google Scholar, 14Hosoi M. Kim S. Tabata T. Nishitani H. Nishizawa Y. Morii H. Murakami K. Yamamoto K. J. Clin. Endocrinol. & Metab. 1992; 74: 680-684PubMed Google Scholar). This finding has raised the question about whether non-renal tissues have the capacity to activate prorenin and thereby carry out the first reaction in the RAS. For these reasons and despite much circumstantial evidence, the functions of tissue RAS remain a matter of conjecture. using transgenic animals, it is possible to test for the biological effect of tissue expression of the RAS by inducing either loss of function or gain of function mutations. Mice lacking all RAS activity have been generated via homologous recombination by insertional mutagenesis of the angiotensinogen gene (the only known substrate for generation of the angiotensin peptides) (15Tanimoto K. Sugiyama F. Goto Y. Ishida J. Takimoto E. Yagami K. Fukamizu A. Murakami K. J. Biol. Chem. 1994; 269: 31334-31337Abstract Full Text PDF PubMed Google Scholar, 16Kim H.S. Krege J.H. Kluckman K.D. Hagaman J.R. Hodgin J.B. Best C.F. Jennette J.C. Coffman T.M. Maeda N. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2735-2739Crossref PubMed Scopus (580) Google Scholar). These mice are hypotensive and exhibit some defects in the development of the kidney, but these experiments shed little light on the normal physiologic functions of tissue RAS. On the other hand, generalized overexpression of components of the RAS in transgenic mice and rats is clearly linked to an increase in blood pressure (17Mullins J.J. Peters J. Ganten D. Nature. 1990; 344: 541-544Crossref PubMed Scopus (807) Google Scholar, 18Ganten D. Wagner J. Zeh K. Bader M. Michel J.B. Paul M. Zimmermann F. Ruf P. Hilgenfeldt U. Ganten U. Kaling M. Bachmann S. Fukamizu A. Mullins J.J. Murakami K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7806-7810Crossref PubMed Scopus (200) Google Scholar, 19Keusch, G., Kurtz, A., Fehr, J., Eckardt, K. U., Frei, D., Bauer, C., and Binswanger, U. (1989) Nephron , 51, Suppl. 1, 29–33.Google Scholar, 20Fukamizu A. Sugimura K. Takimoto E. Sugiyama F. Seo M.-S. Takahashi S. Hatae T. Kajiwara N. Yagami K. Murakami K. J. Biol. Chem. 1993; 268: 11617-11621Abstract Full Text PDF PubMed Google Scholar). To discriminate between circulatory and local effects of transgene expression, it is necessary to target expression of the RAS transgenes to specific tissues. However, this approach is complicated by the fact that all of the RAS components (i.e. angiotensinogen, prorenin, angiotensin-converting enzyme, Ang II receptors, and a prorenin convertase) need to be expressed in the target tissue. In addition, the Ang II generated can elicit biological responses at other an alternative to co-expressing RAS components, we have engineered a fusion protein that leads to the direct release of an Ang II peptide within specific tissues. This peptide is a natural variant of Ang II found in the skin of the Australian frog Crinia georgiana(fsAngII (21Erspamer V. Melchiorri P. Nakajima T. Yasuhara T. ( Basel ). PubMed Scopus Google has been to have activity to that of mammalian Ang II Yasuhara T. Nakajima T. J. Med. Chem. PubMed Scopus Google Scholar). a for proteolytic release of from the fusion protein by the ubiquitous furin T. T. J. M. Murakami K. K. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). In this the and biological activity of the peptide and experiments to test for the effect of local of in Direct injection of the expression vector into the of rat leads to local expression of and of atrial natriuretic peptide expression, a marker of cardiac in cardiac ventricles P. J. Clin. Invest. PubMed Scopus Google Scholar, Deschepper C.F. Am. J. Google Scholar). These results for the first the of using an engineered fusion protein to a peptide local biological activity in have engineered a fusion protein that leads to the direct release of an Ang II within tissues. The angiotensin peptide used in this is released by a by the to the constitutive secretion of the released a mammalian of the is a expressed in all tissues and cell J.A. J. Clin. Invest. PubMed Scopus Google Scholar, J. P.J. J. Biol. 1990; PubMed Scopus Google Scholar, K. M. T. M. A. Murakami K. K. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). for of by furin have been and the amino from to to the has been shown to to the in cells T. Murakami K. K. 1993; PubMed Scopus Google Scholar, S. K. T. Murakami K. K. J. ( ). 1994; PubMed Scopus Google Scholar). II, is an from the skin of the Australian frog (21Erspamer V. Melchiorri P. Nakajima T. Yasuhara T. ( Basel ). PubMed Scopus Google its is a substrate for by furin T. T. J. M. Murakami K. K. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). results that and secretion of from the fusion protein the of a between the and the angiotensin peptide to the The of the prorenin as a by the engineered furin in this peptide V. Baxter J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the in the fusion protein for a of fusion in cell and by the of protein or for the release of peptide is also by the finding that of into of rat leads to intracellular and secretion of results that to the Ang II While the a to that of mammalian Ang II Ang to the is to most of the effects of Ang II P. J. T. Hypertension. PubMed Scopus Google M. P.J. Best C.F. Maeda N. Smithies O. Coffman T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google occurs Ang II However, only of the release of intracellular its of action on target cells is that of Ang II The of these the in biological of Ang II and on and Yasuhara T. Nakajima T. J. Med. Chem. PubMed Scopus Google have that the activity of the in rats that of Ang in injection using rat cardiac for the first that an engineered fusion protein can be to a mature peptide and have local biological effects. are as to the of the cells the peptide in as for either the or angiotensin peptides to a low of expression and/or the secretion of the fusion protein from the However, as the used in expression vector has a cell it be expressed in either cardiac or of rats in blood to or in circulating Ang II between the and In to the of a increase in mRNA in the of the gene expression to the injection the to an increase in to these that the expression of is highly localized to cells the injection These the in results of J. Y. H.S. S. 1993; Full Text PDF PubMed Scopus Google the effects of Ang II in cardiac in that in local overexpression of in the rat leads to a highly localized cardiac we have a expression vector that can as an alternative to co-expressing all the components of the RAS to generate Ang II. This of approach also be the of tissue-specific in transgenic to test the of other peptides on have engineered a fusion protein that leads to the direct release of an Ang II within tissues. The angiotensin peptide used in this is released by a by the to the constitutive secretion of the released a mammalian of the is a expressed in all tissues and cell J.A. J. Clin. Invest. PubMed Scopus Google Scholar, J. P.J. J. Biol. 1990; PubMed Scopus Google Scholar, K. M. T. M. A. Murakami K. K. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). for of by furin have been and the amino from to to the has been shown to to the in cells T. Murakami K. K. 1993; PubMed Scopus Google Scholar, S. K. T. Murakami K. K. J. ( ). 1994; PubMed Scopus Google Scholar). II, is an from the skin of the Australian frog (21Erspamer V. Melchiorri P. Nakajima T. Yasuhara T. ( Basel ). PubMed Scopus Google its is a substrate for by furin T. T. J. M. Murakami K. K. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). results that and secretion of from the fusion protein the of a between the and the angiotensin peptide to the The of the prorenin as a by the engineered furin in this peptide V. Baxter J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the in the fusion protein for a of fusion in cell and by the of protein or for the release of peptide is also by the finding that of into of rat leads to intracellular and secretion of results that to the Ang II While the a to that of mammalian Ang II Ang to the is to most of the effects of Ang II P. J. T. Hypertension. PubMed Scopus Google M. P.J. Best C.F. Maeda N. Smithies O. Coffman T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google occurs Ang II However, only of the release of intracellular its of action on target cells is that of Ang II The of these the in biological of Ang II and on and Yasuhara T. Nakajima T. J. Med. Chem. PubMed Scopus Google have that the activity of the in rats that of Ang II. in injection using rat cardiac for the first that an engineered fusion protein can be to a mature peptide and have local biological effects. are as to the of the cells the peptide in as for either the or angiotensin peptides to a low of expression and/or the secretion of the fusion protein from the However, as the used in expression vector has a cell it be expressed in either cardiac or of rats in blood to or in circulating Ang II between the and In to the of a increase in mRNA in the of the gene expression to the injection the to an increase in to these that the expression of is highly localized to cells the injection These the in results of J. Y. H.S. S. 1993; Full Text PDF PubMed Scopus Google the effects of Ang II in cardiac in that in local overexpression of in the rat leads to a highly localized cardiac In we have a expression vector that can as an alternative to co-expressing all the components of the RAS to generate Ang II. This of approach also be the of tissue-specific in transgenic to test the of other peptides on