Tissue Targeting of Angiotensin Peptides
1997; Elsevier BV; Volume: 272; Issue: 20 Linguagem: Inglês
10.1074/jbc.272.20.12994
ISSN1083-351X
AutoresDanielle Méthot, Margot C. LaPointe, Rhian M. Touyz, Xiao-Ping Yang, Oscar A. Carretero, Christian F. Deschepper, Ernesto L. Schiffrin, Gaétan Thibault, Timothy L. Reudelhuber,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoAngiotensin 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. Endean R. Experientia ( Basel ). 1979; 35: 1132-1133Crossref PubMed Scopus (35) Google Scholar)) which has been reported to have pressor activity similar to that of mammalian Ang II (22Khosla M.C. Bumpus F.M. Yasuhara T. Nakajima T. J. Med. Chem. 1981; 24: 885-887Crossref PubMed Scopus (10) Google Scholar). Its amino-terminal alanine residue presents a favorable P1′ cleavage site for proteolytic release of fsAngII from the fusion protein by the ubiquitous protease furin (23Watanabe T. Nakagawa T. Ikemizu J. Nagahama M. Murakami K. Nakayama K. J. Biol. Chem. 1992; 267: 8270-8274Abstract Full Text PDF PubMed Google Scholar). In this study, the receptor binding and biological activity of the fsAngII peptide were characterized, and experiments were performed to test for the effect of local over-expression of fsAngII in vivo. Direct injection of the expression vector into the apex of rat hearts leads to local expression of fsAngII and induction of atrial natriuretic peptide (ANP) expression, a biochemical molecular marker of cardiac hypertrophy in adult cardiac ventricles (24Wei Y.F. Rodi C.P. Day M.L. Wiegand R.C. Needleman L.D. Cole B.R. Needleman P. J. Clin. Invest. 1987; 79: 1325-1329Crossref PubMed Scopus (100) Google Scholar, 25Wu J.P. Deschepper C.F. Gardner D.G. Am. J. Physiol. 1988; 255: E388-E396PubMed Google Scholar). These results demonstrate for the first time the feasibility of using an engineered fusion protein to deliver a peptide with local biological activity in whole animals.DISCUSSIONWe have engineered a fusion protein that leads to the direct release of an Ang II analog within transfected tissues. The angiotensin peptide used in this study is released by a single cleavage, effected by the processing protease furin, leading to the constitutive secretion of the released peptide. Furin, a mammalian homolog of the yeast precursor-processing Kex2 endoprotease, is a Golgi-anchored convertase expressed in all examined tissues and cell lines (42Schalken J.A. Roebroek A.J. Oomen P.P. Wagenaar S.S. Debruyne F.M. Bloemers H.P. Van de Ven W.J. J. Clin. Invest. 1987; 80: 1545-1549Crossref PubMed Scopus (124) Google Scholar, 43Bresnahan P.A. Leduc R. Thomas L. Thorner J. Gibson H.L. Brake A.J. Barr P.J. Thomas G. J. Cell Biol. 1990; 111: 2851-2859Crossref PubMed Scopus (288) Google Scholar, 44Hatsuzawa K. Hosaka M. Nakagawa T. Nagase M. Shoda A. Murakami K. Nakayama K. J. Biol. Chem. 1990; 265: 22075-22078Abstract Full Text PDF PubMed Google Scholar). Sequence requirements for efficient processing of precursors by furin have been extensively studied, and the RXRXKR amino acid combination located from position −6 to −1 relative to the cleavage site has been shown to lead to the higher cleavage efficiency in cultured cells (29Watanabe T. Murakami K. Nakayama K. FEBS Lett. 1993; 320: 215-218Crossref PubMed Scopus (56) Google Scholar, 30Takahashi S. Hatsuzawa K. Watanabe T. Murakami K. Nakayama K. J. Biochem. ( Tokyo ). 1994; 116: 47-52Crossref PubMed Scopus (37) Google Scholar). fsAngII, Ala-Pro-Gly-[Ile3,Val5]Ang II, is an undecapeptide isolated from the skin of the Australian frog C. georgiana (21Erspamer V. Melchiorri P. Nakajima T. Yasuhara T. Endean R. Experientia ( Basel ). 1979; 35: 1132-1133Crossref PubMed Scopus (35) Google Scholar), which was chosen because its amino-terminal alanine residue is a good substrate for efficient processing by furin (23Watanabe T. Nakagawa T. Ikemizu J. Nagahama M. Murakami K. Nakayama K. J. Biol. Chem. 1992; 267: 8270-8274Abstract Full Text PDF PubMed Google Scholar). Our results show that efficient cleavage and secretion of fsAngII from the fusion protein requires the presence of a molecular spacer between the Ig fragment and the angiotensin peptide presumably due to steric constraints near the cleavage site. The choice of the prorenin prosegment as a spacer was dictated by our previous success with the engineered furin cleavage in this peptide (27Brechler V. Chu W.N. Baxter J.D. Thibault G. Reudelhuber T.L. J. Biol. Chem. 1996; 271: 20636-20640Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The presence of the Ig fragment in the fusion protein allows for a rapid assessment of cleavage efficiencies with various peptide-containing fusion proteins in cell cultures (Fig. 4 and data not shown) by the simple use of protein A- or G-coupled matrices. Evidence for the cleavage-mediated release of fsAngII peptide is also provided by the finding that introduction of pIgPfsAngII into primary cultures of rat neonatal ventricular myocytes leads to intracellular accumulation and secretion of immunoreactive fsAngII (Table I).Our results demonstrate that fsAngII binds to both the AT1and AT2 Ang II receptor subtypes. While fsAngII binds the AT2 receptor with a similar affinity to that of mammalian Ang II (native Ang II), binding to the AT1 receptor, which is thought to mediate most of the cardiovascular effects of Ang II (34Touyz R.M. Sventek P. Lariviere R. Thibault G. Fareh J. Reudelhuber T. Schiffrin E.L. Hypertension. 1996; 27: 1090-1096Crossref PubMed Scopus (43) Google Scholar,45Ito M. Oliverio M.I. Mannon P.J. Best C.F. Maeda N. Smithies O. Coffman T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3521-3525Crossref PubMed Scopus (545) Google Scholar), occurs with 40-fold lower affinity than native Ang II (Fig.2 A). However, even though fsAngII binding elicits only approximately 65% of the AT1 receptor-mediated release of intracellular calcium, its duration of action on target cells is significantly longer than that of native Ang II (Fig. 3). The combination of these properties may explain the apparent similarities in effective biological concentrations of Ang II and fsAngII on transfected cells. Indeed, Khosla and co-workers (22Khosla M.C. Bumpus F.M. Yasuhara T. Nakajima T. J. Med. Chem. 1981; 24: 885-887Crossref PubMed Scopus (10) Google Scholar) have previously reported that the pressor activity of the synthetic fsAngII in rats was 90.6 ± 5.0% that of human Ang II.Our in vivo injection experiments, using pIgPfsAngII-injected rat cardiac ventricles, demonstrate for the first time that an engineered fusion protein precursor (Ig-prosegment-fsAngII) can be processed to a mature peptide (fsAngII)in vivo and have local biological effects. We are unsure as to the origin of the cells secreting the fsAngII peptide in injected hearts as immunostaining for either the Ig or angiotensin peptides was inconclusive (perhaps due to a low level of expression and/or the rapid secretion of the fusion protein from the cells). However, as the viral promoter/enhancer used in our expression vector has a broad cell specificity, it could conceivably be expressed in either cardiac myocytes or fibroblasts. Analyses of injected rats revealed no significant differences in blood pressure, total heart weight to body weight ratios (Table II), or in circulating Ang II levels (data not shown) between the control and pIgPfsAngII-expressing groups. In contrast to the absence of systemic fsAngII effects, fsAngII induced a significant increase in ANP mRNA levels in the injected portion of the heart (Fig. 5). Enhanced ANP gene expression was not detected distal to the injection site. Coupled with the failure to detect an increase in heart to body weight ratios, these data suggest that the expression of fsAngII is highly localized to cells along the injection site. These data support the in vitro results of Sadoshima et al. (46Sadoshima J. Xu Y. Slayter H.S. Izumo S. Cell. 1993; 75: 977-984Abstract Full Text PDF PubMed Scopus (1160) Google Scholar) who demonstrated the autocrine effects of Ang II in cardiac myocyte hypertrophy in vitroand demonstrate that in vivo local overexpression of fsAngII in the rat heart leads to a highly localized cardiac hypertrophic phenotype.In conclusion, we have described a novel expression vector that can serve as an alternative to co-expressing all the components of the RAS to generate Ang II. This type of approach could also be extended with the use of tissue-specific genes in transgenic animals to test the importance of other bioactive peptides on organ physiology. 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. Endean R. Experientia ( Basel ). 1979; 35: 1132-1133Crossref PubMed Scopus (35) Google Scholar)) which has been reported to have pressor activity similar to that of mammalian Ang II (22Khosla M.C. Bumpus F.M. Yasuhara T. Nakajima T. J. Med. Chem. 1981; 24: 885-887Crossref PubMed Scopus (10) Google Scholar). Its amino-terminal alanine residue presents a favorable P1′ cleavage site for proteolytic release of fsAngII from the fusion protein by the ubiquitous protease furin (23Watanabe T. Nakagawa T. Ikemizu J. Nagahama M. Murakami K. Nakayama K. J. Biol. Chem. 1992; 267: 8270-8274Abstract Full Text PDF PubMed Google Scholar). In this study, the receptor binding and biological activity of the fsAngII peptide were characterized, and experiments were performed to test for the effect of local over-expression of fsAngII in vivo. Direct injection of the expression vector into the apex of rat hearts leads to local expression of fsAngII and induction of atrial natriuretic peptide (ANP) expression, a biochemical molecular marker of cardiac hypertrophy in adult cardiac ventricles (24Wei Y.F. Rodi C.P. Day M.L. Wiegand R.C. Needleman L.D. Cole B.R. Needleman P. J. Clin. Invest. 1987; 79: 1325-1329Crossref PubMed Scopus (100) Google Scholar, 25Wu J.P. Deschepper C.F. Gardner D.G. Am. J. Physiol. 1988; 255: E388-E396PubMed Google Scholar). These results demonstrate for the first time the feasibility of using an engineered fusion protein to deliver a peptide with local biological activity in whole animals. DISCUSSIONWe have engineered a fusion protein that leads to the direct release of an Ang II analog within transfected tissues. The angiotensin peptide used in this study is released by a single cleavage, effected by the processing protease furin, leading to the constitutive secretion of the released peptide. Furin, a mammalian homolog of the yeast precursor-processing Kex2 endoprotease, is a Golgi-anchored convertase expressed in all examined tissues and cell lines (42Schalken J.A. Roebroek A.J. Oomen P.P. Wagenaar S.S. Debruyne F.M. Bloemers H.P. Van de Ven W.J. J. Clin. Invest. 1987; 80: 1545-1549Crossref PubMed Scopus (124) Google Scholar, 43Bresnahan P.A. Leduc R. Thomas L. Thorner J. Gibson H.L. Brake A.J. Barr P.J. Thomas G. J. Cell Biol. 1990; 111: 2851-2859Crossref PubMed Scopus (288) Google Scholar, 44Hatsuzawa K. Hosaka M. Nakagawa T. Nagase M. Shoda A. Murakami K. Nakayama K. J. Biol. Chem. 1990; 265: 22075-22078Abstract Full Text PDF PubMed Google Scholar). Sequence requirements for efficient processing of precursors by furin have been extensively studied, and the RXRXKR amino acid combination located from position −6 to −1 relative to the cleavage site has been shown to lead to the higher cleavage efficiency in cultured cells (29Watanabe T. Murakami K. Nakayama K. FEBS Lett. 1993; 320: 215-218Crossref PubMed Scopus (56) Google Scholar, 30Takahashi S. Hatsuzawa K. Watanabe T. Murakami K. Nakayama K. J. Biochem. ( Tokyo ). 1994; 116: 47-52Crossref PubMed Scopus (37) Google Scholar). fsAngII, Ala-Pro-Gly-[Ile3,Val5]Ang II, is an undecapeptide isolated from the skin of the Australian frog C. georgiana (21Erspamer V. Melchiorri P. Nakajima T. Yasuhara T. Endean R. Experientia ( Basel ). 1979; 35: 1132-1133Crossref PubMed Scopus (35) Google Scholar), which was chosen because its amino-terminal alanine residue is a good substrate for efficient processing by furin (23Watanabe T. Nakagawa T. Ikemizu J. Nagahama M. Murakami K. Nakayama K. J. Biol. Chem. 1992; 267: 8270-8274Abstract Full Text PDF PubMed Google Scholar). Our results show that efficient cleavage and secretion of fsAngII from the fusion protein requires the presence of a molecular spacer between the Ig fragment and the angiotensin peptide presumably due to steric constraints near the cleavage site. The choice of the prorenin prosegment as a spacer was dictated by our previous success with the engineered furin cleavage in this peptide (27Brechler V. Chu W.N. Baxter J.D. Thibault G. Reudelhuber T.L. J. Biol. Chem. 1996; 271: 20636-20640Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The presence of the Ig fragment in the fusion protein allows for a rapid assessment of cleavage efficiencies with various peptide-containing fusion proteins in cell cultures (Fig. 4 and data not shown) by the simple use of protein A- or G-coupled matrices. Evidence for the cleavage-mediated release of fsAngII peptide is also provided by the finding that introduction of pIgPfsAngII into primary cultures of rat neonatal ventricular myocytes leads to intracellular accumulation and secretion of immunoreactive fsAngII (Table I).Our results demonstrate that fsAngII binds to both the AT1and AT2 Ang II receptor subtypes. While fsAngII binds the AT2 receptor with a similar affinity to that of mammalian Ang II (native Ang II), binding to the AT1 receptor, which is thought to mediate most of the cardiovascular effects of Ang II (34Touyz R.M. Sventek P. Lariviere R. Thibault G. Fareh J. Reudelhuber T. Schiffrin E.L. Hypertension. 1996; 27: 1090-1096Crossref PubMed Scopus (43) Google Scholar,45Ito M. Oliverio M.I. Mannon P.J. Best C.F. Maeda N. Smithies O. Coffman T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3521-3525Crossref PubMed Scopus (545) Google Scholar), occurs with 40-fold lower affinity than native Ang II (Fig.2 A). However, even though fsAngII binding elicits only approximately 65% of the AT1 receptor-mediated release of intracellular calcium, its duration of action on target cells is significantly longer than that of native Ang II (Fig. 3). The combination of these properties may explain the apparent similarities in effective biological concentrations of Ang II and fsAngII on transfected cells. Indeed, Khosla and co-workers (22Khosla M.C. Bumpus F.M. Yasuhara T. Nakajima T. J. Med. Chem. 1981; 24: 885-887Crossref PubMed Scopus (10) Google Scholar) have previously reported that the pressor activity of the synthetic fsAngII in rats was 90.6 ± 5.0% that of human Ang II.Our in vivo injection experiments, using pIgPfsAngII-injected rat cardiac ventricles, demonstrate for the first time that an engineered fusion protein precursor (Ig-prosegment-fsAngII) can be processed to a mature peptide (fsAngII)in vivo and have local biological effects. We are unsure as to the origin of the cells secreting the fsAngII peptide in injected hearts as immunostaining for either the Ig or angiotensin peptides was inconclusive (perhaps due to a low level of expression and/or the rapid secretion of the fusion protein from the cells). However, as the viral promoter/enhancer used in our expression vector has a broad cell specificity, it could conceivably be expressed in either cardiac myocytes or fibroblasts. Analyses of injected rats revealed no significant differences in blood pressure, total heart weight to body weight ratios (Table II), or in circulating Ang II levels (data not shown) between the control and pIgPfsAngII-expressing groups. In contrast to the absence of systemic fsAngII effects, fsAngII induced a significant increase in ANP mRNA levels in the injected portion of the heart (Fig. 5). Enhanced ANP gene expression was not detected distal to the injection site. Coupled with the failure to detect an increase in heart to body weight ratios, these data suggest that the expression of fsAngII is highly localized to cells along the injection site. These data support the in vitro results of Sadoshima et al. (46Sadoshima J. Xu Y. Slayter H.S. Izumo S. Cell. 1993; 75: 977-984Abstract Full Text PDF PubMed Scopus (1160) Google Scholar) who demonstrated the autocrine effects of Ang II in cardiac myocyte hypertrophy in vitroand demonstrate that in vivo local overexpression of fsAngII in the rat heart leads to a highly localized cardiac hypertrophic phenotype.In conclusion, we have described a novel expression vector that can serve as an alternative to co-expressing all the components of the RAS to generate Ang II. This type of approach could also be extended with the use of tissue-specific genes in transgenic animals to test the importance of other bioactive peptides on organ physiology. We have engineered a fusion protein that leads to the direct release of an Ang II analog within transfected tissues. The angiotensin peptide used in this study is released by a single cleavage, effected by the processing protease furin, leading to the constitutive secretion of the released peptide. Furin, a mammalian homolog of the yeast precursor-processing Kex2 endoprotease, is a Golgi-anchored convertase expressed in all examined tissues and cell lines (42Schalken J.A. Roebroek A.J. Oomen P.P. Wagenaar S.S. Debruyne F.M. Bloemers H.P. Van de Ven W.J. J. Clin. Invest. 1987; 80: 1545-1549Crossref PubMed Scopus (124) Google Scholar, 43Bresnahan P.A. Leduc R. Thomas L. Thorner J. Gibson H.L. Brake A.J. Barr P.J. Thomas G. J. Cell Biol. 1990; 111: 2851-2859Crossref PubMed Scopus (288) Google Scholar, 44Hatsuzawa K. Hosaka M. Nakagawa T. Nagase M. Shoda A. Murakami K. Nakayama K. J. Biol. Chem. 1990; 265: 22075-22078Abstract Full Text PDF PubMed Google Scholar). Sequence requirements for efficient processing of precursors by furin have been extensively studied, and the RXRXKR amino acid combination located from position −6 to −1 relative to the cleavage site has been shown to lead to the higher cleavage efficiency in cultured cells (29Watanabe T. Murakami K. Nakayama K. FEBS Lett. 1993; 320: 215-218Crossref PubMed Scopus (56) Google Scholar, 30Takahashi S. Hatsuzawa K. Watanabe T. Murakami K. Nakayama K. J. Biochem. ( Tokyo ). 1994; 116: 47-52Crossref PubMed Scopus (37) Google Scholar). fsAngII, Ala-Pro-Gly-[Ile3,Val5]Ang II, is an undecapeptide isolated from the skin of the Australian frog C. georgiana (21Erspamer V. Melchiorri P. Nakajima T. Yasuhara T. Endean R. Experientia ( Basel ). 1979; 35: 1132-1133Crossref PubMed Scopus (35) Google Scholar), which was chosen because its amino-terminal alanine residue is a good substrate for efficient processing by furin (23Watanabe T. Nakagawa T. Ikemizu J. Nagahama M. Murakami K. Nakayama K. J. Biol. Chem. 1992; 267: 8270-8274Abstract Full Text PDF PubMed Google Scholar). Our results show that efficient cleavage and secretion of fsAngII from the fusion protein requires the presence of a molecular spacer between the Ig fragment and the angiotensin peptide presumably due to steric constraints near the cleavage site. The choice of the prorenin prosegment as a spacer was dictated by our previous success with the engineered furin cleavage in this peptide (27Brechler V. Chu W.N. Baxter J.D. Thibault G. Reudelhuber T.L. J. Biol. Chem. 1996; 271: 20636-20640Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The presence of the Ig fragment in the fusion protein allows for a rapid assessment of cleavage efficiencies with various peptide-containing fusion proteins in cell cultures (Fig. 4 and data not shown) by the simple use of protein A- or G-coupled matrices. Evidence for the cleavage-mediated release of fsAngII peptide is also provided by the finding that introduction of pIgPfsAngII into primary cultures of rat neonatal ventricular myocytes leads to intracellular accumulation and secretion of immunoreactive fsAngII (Table I). Our results demonstrate that fsAngII binds to both the AT1and AT2 Ang II receptor subtypes. While fsAngII binds the AT2 receptor with a similar affinity to that of mammalian Ang II (native Ang II), binding to the AT1 receptor, which is thought to mediate most of the cardiovascular effects of Ang II (34Touyz R.M. Sventek P. Lariviere R. Thibault G. Fareh J. Reudelhuber T. Schiffrin E.L. Hypertension. 1996; 27: 1090-1096Crossref PubMed Scopus (43) Google Scholar,45Ito M. Oliverio M.I. Mannon P.J. Best C.F. Maeda N. Smithies O. Coffman T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3521-3525Crossref PubMed Scopus (545) Google Scholar), occurs with 40-fold lower affinity than native Ang II (Fig.2 A). However, even though fsAngII binding elicits only approximately 65% of the AT1 receptor-mediated release of intracellular calcium, its duration of action on target cells is significantly longer than that of native Ang II (Fig. 3). The combination of these properties may explain the apparent similarities in effective biological concentrations of Ang II and fsAngII on transfected cells. Indeed, Khosla and co-workers (22Khosla M.C. Bumpus F.M. Yasuhara T. Nakajima T. J. Med. Chem. 1981; 24: 885-887Crossref PubMed Scopus (10) Google Scholar) have previously reported that the pressor activity of the synthetic fsAngII in rats was 90.6 ± 5.0% that of human Ang II. Our in vivo injection experiments, using pIgPfsAngII-injected rat cardiac ventricles, demonstrate for the first time that an engineered fusion protein precursor (Ig-prosegment-fsAngII) can be processed to a mature peptide (fsAngII)in vivo and have local biological effects. We are unsure as to the origin of the cells secreting the fsAngII peptide in injected hearts as immunostaining for either the Ig or angiotensin peptides was inconclusive (perhaps due to a low level of expression and/or the rapid secretion of the fusion protein from the cells). However, as the viral promoter/enhancer used in our expression vector has a broad cell specificity, it could conceivably be expressed in either cardiac myocytes or fibroblasts. Analyses of injected rats revealed no significant differences in blood pressure, total heart weight to body weight ratios (Table II), or in circulating Ang II levels (data not shown) between the control and pIgPfsAngII-expressing groups. In contrast to the absence of systemic fsAngII effects, fsAngII induced a significant increase in ANP mRNA levels in the injected portion of the heart (Fig. 5). Enhanced ANP gene expression was not detected distal to the injection site. Coupled with the failure to detect an increase in heart to body weight ratios, these data suggest that the expression of fsAngII is highly localized to cells along the injection site. These data support the in vitro results of Sadoshima et al. (46Sadoshima J. Xu Y. Slayter H.S. Izumo S. Cell. 1993; 75: 977-984Abstract Full Text PDF PubMed Scopus (1160) Google Scholar) who demonstrated the autocrine effects of Ang II in cardiac myocyte hypertrophy in vitroand demonstrate that in vivo local overexpression of fsAngII in the rat heart leads to a highly localized cardiac hypertrophic phenotype. In conclusion, we have described a novel expression vector that can serve as an alternative to co-expressing all the components of the RAS to generate Ang II. This type of approach could also be extended with the use of tissue-specific genes in transgenic animals to test the importance of other bioactive peptides on organ physiology.
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