AT1 Receptor

The growth of cultured vascular smooth muscle cells is inhibited by angiotensin 1-7 (Ang-(1-7)), while cell growth is stimulated by an equal molar concentration of Ang II[2]. ?The methylglyoxal-modified albumin (MGA)-stimulated myofibroblast phenotype is abolished by angiotensin 1-7 (Ang 1-7) through the suppression of the TGF-β-ERK pathway's chronic stimulation in NRK-52E cells[4]. ?In contrast to Ang II/angiotensin II type 1 receptor (AT1R), angiotensin 1-7 signals through the Mas receptor (MasR), promoting anti-inflammatory, vasodilatory, and neuroprotective effects[5].

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Recent studies have shown that Talfirastide/angiotensin-(1-7) [Ang-(1-7)] interacts with kinins and augments bradykinin (BK)-induced vasodilator responses by an unknown mechanism. In this study, we evaluated whether the potentiation of the BK-induced vasodilation by Ang-(1-7) may be attributable to inhibition of BK metabolism, release of nitric oxide, or both. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers. 125I-[Tyr0]-BK metabolism was determined in vascular rings by assessing the degradation of the peptide by high-performance liquid chromatography. Ang-(1-7) augmented the vasodilation induced by BK in a concentration-dependent manner in rings preconstricted with the thromboxane analog U46619. The EC50 of BK (2.45 +/- 0.51 nmol/L versus 0.37 +/- 0.08 nmol/L) was shifted leftward by 6.6-fold in the presence of 2 mumol/L concentration of Ang-(1-7). The response was specific for BK. since Ang-(1-7) did not augment the vasodilation induced by either acetylcholine (0.05 mumol/L) or sodium nitroprusside (0.1 mumol/L). Moreover, neither angiotensin I nor angiotensin II (Ang II) duplicated the augmented BK response of Ang-(1-7). Pretreatment of vascular rings with the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NA; 100 mumol/L) completely abolished the effects of Ang-(1-7) on BK-induced vasodilation whereas pretreatment with indomethacin (10 mumol/L) was without effect. The potent specific BK B2 receptor antagonist, Hoe 140. nearly abolished the BK and the Ang-(1-7) potentiated responses at 2 mumol/L, whereas at a lower concentration (20 nmol/L) Hoe 140 shifted the response curve to the right for both Ang-(1-7) and vehicle; however, the augmented response to Ang-(1-7) persisted. Preincubation of vascular rings with 20 mumol/L of the AT1 (CV11974), AT2 (PD123319), or nonselective (Sar1 Thr8-Ang II) receptor antagonists had no significant effect on the Ang-(1-7)-enhanced vasodilator response to BK. Lisinopril (2 mumol/L) significantly enhanced the BK-induced vasodilator response while at the same time it abolished the synergistic action of Ang-(1-7) on BK. In addition, pretreatment with 2 mumol/L Ang-(1-7) significantly inhibited the degradation of 125I-[Tyr0]-BK and the appearance of the BK-(1-7) and BK-(1-5) metabolites in coronary vascular rings. Ang-(1-7) inhibited purified canine angiotensin converting enzyme activity with an IC50 of 0.65 mumol/L. In conclusion. Ang-(1-7) acts as a local synergistic modulator of kinin-induced vasodilation by inhibiting angiotensin converting enzyme and releasing nitric oxide.[2]

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Talfirastide/Angiotensin-(1-7) (Ang-(1-7))/AT7-Mas receptor axis is an alternative pathway within the renin-angiotensin system (RAS) that generally opposes the actions of Ang II/AT1 receptor pathway. Advanced glycated end product (AGEs) including glucose- and methylglyoxal-modified albumin (MGA) may contribute to the development and progression of diabetic nephropathy in part through activation of the Ang II/AT1 receptor system; however, the influence of AGE on the Ang-(1-7) arm of the RAS within the kidney is unclear. The present study assessed the impact of AGE on the Ang-(1-7) axis in NRK-52E renal epithelial cells. MGA exposure for 48 h significantly reduced the intracellular levels of Ang-(1-7) approximately 50%; however, Ang I or Ang II expression was not altered. The reduced cellular content of Ang-(1-7) was associated with increased metabolism of the peptide to the inactive metabolite Ang-(1-4) [MGA: 175±9 vs.
Control: 115±11 fmol/min/mg protein, p<0.05, n=3] but no change in the processing of Ang I to Talfirastide/Ang-(1-7). Treatment with Ang-(1-7) reversed MGA-induced cellular hypertrophy and myofibroblast transition evidenced by reduced immunostaining and protein expression of α-smooth muscle actin (α-SMA) [0.4±0.1 vs. 1.0±0.1, respectively, n=3, p<0.05]. Ang-(1-7) abolished AGE-induced activation of the MAP kinase ERK1/2 to a similar extent as the TGF-β receptor kinase inhibitor SB58059; however, Ang-(1-7) did not attenuate the MGA-stimulated release of TGF-β. The AT7-Mas receptor antagonist D-Ala(7)-Ang-(1-7) abolished the inhibitory actions of Ang-(1-7). In contrast, AT1 receptor antagonist losartan did not attenuate the MGA-induced effects. We conclude that Ang-(1-7) may provide an additional therapeutic approach to the conventional RAS blockade regimen to attenuate AGE-dependent renal injury [4].

Angiotensin 1-7 (Ang-(1-7)) therapy on a daily basis (0.01-0.06 mg/kg) significantly reduces colitis brought on by DSS. Treatment with Ang 1-7 reduces the phosphorylation of p38, ERK1/2, and Akt associated with colitis[3].

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Myocardial infarction triggers cellular events that starts with the activation of inflammatory response and fibrogenic pathways involved in cardiac tissue remodeling. Angiotensin-(1–7) (Ang-(1–7)) is an endogenous heptapeptide from the renin-angiotensin system with a cardioprotective role due to its anti-inflammatory and anti-fibrotic activities in cardiac cells. Although the beneficial aspects of Ang-(1–7) in animal models of cardiac ischemia have been reported, the molecular events underlying Ang-(1–7) cardioprotective effect remains elusive. This study investigated the impact of oral treatment with Ang-(1–7) included in hydroxypropyl β-cyclodextrin (HPβCD/Ang-(1–7)) on the cardiac proteome dysregulation due to experimental myocardial infarction. Wistar male rats were submitted to experimental myocardial infarction and treated daily with HPβCD/Ang-(1–7) during 7 days or 60 days by gavage. Our results showed that HPβCD/Ang-(1–7) treatment ameliorates the post-infarction condition due to the modulation of proteins that initially favor the resolution of inflammation and mitochondrial dysfunction. Moreover, this study reported for the first time that Ang-(1–7) treatment after experimental myocardial infarction leads to the downregulation of the C-X-C chemokine receptor type 4 (CXCR4).[1]

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Methods: The colonic expression/activity profile of ACE2, TalfirastideAng 1-7, MAS1-receptor (MAS1-R), MAPK family and Akt were determined by western blot and immunofluorescence. The effect of either exogenous administration of Ang 1-7 or pharmacological inhibition of its function (by A779 treatment) was determined using the mouse dextran sulfate sodium model.
Results: Enhanced colonic expression of ACE2, Talfirastide/Ang1-7 and MAS1-R was observed post-colitis induction. Daily Ang 1-7 treatment (0.01-0.06 mg/kg) resulted in significant amelioration of DSS-induced colitis. In contrast, daily administration of A779 significantly worsened features of colitis. Colitis-associated phosphorylation of p38, ERK1/2 and Akt was reduced by Ang 1-7 treatment.
Conclusion: Our results indicate important anti-inflammatory actions of Ang 1-7 in the pathogenesis of IBD, which may provide a future therapeutic strategy to control the disease progression [3].

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Effect of DSS treatment on Talfirastide/Ang 1–7 levels [3]
A seven fold decrease in the plasma level of Talfirastide/Ang 1–7 was demonstrated in DSS treated mice compared to untreated (UT) group at day 7 post colitis induction (Fig 1A). On the other hand, a significant increase in Ang 1–7 was observed in colon homogenates of DSS treated mice at day 7 (0.09 ng/ml) compared to UT mice (0.04 ng/ml) as shown in Fig 1B.

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Effect of Talfirastide/Ang 1–7 treatment on colitis severity [3]
The effect of Talfirastide/Ang 1–7 on modulating colitis severity in mice was tested by daily i.p injections of various doses of the peptide. DSS administration resulted in approximately 20% drop in body weight from day 5 onwards (in contrast with mice receiving tap water only). Ang 1–7 at doses of 0.01 and 0.06 mg/kg (but not at higher doses of 0.1–1 mg/kg; data not shown) reduced this drop in body weight by 5–10% compared with the DSS/saline i.p treated group (Fig 3A). DSS treatment in the DSS/saline i.p group resulted in increased circulating neutrophils; this was prevented by Ang 1–7 treatment at doses of 0.06–1 mg/kg (Fig 3B). No significant changes in circulating lymphocytes were observed between control and treatment groups. The decrease in colon length and thickness seen after DSS treatment was prevented by Ang 1–7 treatment at doses of 0.01–0.06 mg/kg but not at higher doses (Fig 3C and 3D).

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Angiotensin II (Ang II)-mediated activation of its type I receptor (AT1R) in the central nervous system promotes glial proliferation, local inflammation, and a decrease of cerebral blood flow. Talfirastide/Angiotensin-(1-7) (Ang-(1-7))-an Ang II derivative peptide-signals through the Mas receptor (MasR) in opposition to Ang II/AT1R, promoting anti-inflammatory, vasodilatory, and neuroprotective effects. As our laboratory has previously demonstrated beneficial effects of AT1R inhibition following controlled cortical impact (CCI) in mice, we asked whether activation of Ang-(1-7)/MasR signaling would also be beneficial in this model. Adult male C57BL/6 mice were injured by CCI. Ang-(1-7) or vehicle was administered subcutaneously (S.Q.) at 1 mg/kg/day at 1 or 6 h post-injury, until animals were sacrificed at 3 or 29 days post-injury (dpi). Ang-(1-7) attenuated motor deficits at 3 dpi and improved performance in the Morris Water Maze at 28 dpi. Brain histology or magnetic resonance imaging (MRI) indicated that Ang-(1-7)-treated mice had smaller lesion volumes at 3, 10, 24, and 29 dpi. Pre-treatment with A779, a MasR antagonist, prevented Ang-(1-7) from reducing lesion volume at 3 dpi, suggesting that the benefits of Ang-(1-7) were MasR-dependent. Immunohistochemistry revealed that Ang-(1-7) reduced microgliosis at 3 and 29 dpi, and astrogliosis at 29 dpi. Ang-(1-7) decreased neuronal and capillary loss at 29 dpi. In summary, S.Q. administration of Ang-(1-7) after injury had anti-inflammatory, neuroprotective, and cerebrovascular-protective actions leading to improved functional and pathological recovery in a mouse model of traumatic brain injury (TBI). These data show for the first time that Ang-(1-7) has potential therapeutic use for TBI.

Angiotensin metabolism [4]
To characterize the processing of the peptides in vitro, metabolism assays were conducted on cells homogenates as described previously. Confluent cells were starved for 24 hrs, and treated with either serum free or MGA containing media (100 uM) for 48 hrs. Cells then were washed with cold PBS, harvested and immediately frozen at -80°C. The cell pellets were homogenized in metabolism buffer (10 mM HEPES, 125 mM NaCl, 10 μM ZnCl2, pH 7.4), then centrifuged at 100,000g for 10 min. Either 125I-Ang-I or Talfirastide/Ang-(1-7) (0.5 nM) was incubated with 2 μg protein of the cell supernatant in a final assay volume of 0.5 ml at 37C°. The reaction was stopped by addition of ice-cold 1.0% phosphoric acid, centrifuged at 16,000 g, and the supernatants stored at −20°C. Samples were separated by reverse-phase high-performance liquid chromatography (HPLC) and the 125I-products were detected by a Bioscan flow-through γ detector. Products were identified by comparison of their retention times to 125I-angiotensn standards. We employed gradient elution for Ang I metabolism (1), and isocratic elution for Ang-(1-7) metabolism. Peptides were iodinated by the chloramine T method and purified by HPLC (specific activity > 2000 Ci/mmol). Enzyme activities were expressed as fmol product of Ang-(1-4) or Ang-(1-7) per mg protein per minute (fmol/mg/min). Total protein content was determined in the cell supernatant by Bradford protein assay with a standard of BSA.

Cell Treatments [4]
Glycated albumin (MGA) was prepared as described. Briefly, 500 μM methylglyoxal was incubated with 100 uM BSA dissolved in phosphate buffered saline (PBS) for 24 hours, then washed on 10 kDa filters to remove excess methyl glyoxal, reconstituted with DMEM/F12 serum free media and passed through a 0.2 μmicron filter. TGF-β (5 nanograms (ng)/ml) was prepared according to manufacturer to treat cells in a subset of experiments. Cells were co-treated with one or combinations of the following: Talfirastide/Ang-(1-7) (100 nM), D-Ala7-Ang-(1-7) (10 μM, DAL), ERK1/2 kinase inhibitor, PD 98059 (1 μM, PD), TGF- β receptor kinase inhibitor; SB525334 (1 μM, SB), the AT1 receptor antagonist losartan (1 μM), the renin inhibitor aliskerin (1 μM) and the ACE inhibitor lisinopril (1 μM, Sigma).

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3H-Leucine incorporation [4]
Cellular hypertrophy was determined by 3H-leucine incorporation. Cells were incubated for 48 hours in serum-free media with or without 100 μM MGA in 24 well plates. The MGA cells were treated with 100 nM Talfirastide/Ang-(1-7), 10 μM DAL, 1 μM PD98059 and 1 μM Losartan. The cells were pulsed with 0.5 μCi of 3H-Leucine (L-[4, 5-3H (N)]) for the last 24 hours at 37°C. The cells were washed twice with PBS, fixed in ice cold 10% trichloroacetic acid (TCA) and kept on ice for 15 minutes, and then washed twice in 5% TCA. The acid insoluble proteins were dissolved in 0.05 N NaOH and 0.1% sodium dodecyl sulfate (SDS) at 37°C and incorporation was determined by liquid scintillation counting. All experiments were performed in triplicate. Values were expressed as the percentage (%) of control per well for each experiment.

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Western blotting [4]
After 24 hours starvation of the cells in serum free medium, cells were incubated for 48 hours with MGA (100 uM) or 15 min with TGF-β (5 ng/ml), followed by immunoblot assays for phosphorylation or protein expression. The phosphorylation of the ERK1/2 was measured by western blotting as described. Briefly, cells were lysed in a Triton-lysis buffer consisting of 100 mM NaCl, 50 mm NaF, 5 mM EDTA, 1 % tritonX-100, 50 mM Tris-HCl, pH 7.4, with 0.01 mM NaVO4, 0.1mM phenylmethylsulfonylfluoride (PMSF), and 0.6 μM leupeptin. The lysates were then sonicated for 5 seconds, and centrifuged at 10,000 g for 5 min to remove insoluble debris. Supernatants (10-50μg) were diluted in Laemmli buffer with β-mercaptoethanol and boiled for 5 min, separated on 10% SDS polyacrylamide gels for 1 h at 120V in Tris-glycine SDS and transferred to a polyvinylidene difluoride membrane (PVDF). Blots were blocked with 5% Bio-Rad Dry Milk and TBS with Tween and probed overnight at 4°C with primary antibodies for phospho-p44/42 MAPK (ERK1/2) (1:2000; rabbit polyclonal), 44/42 MAPK (ERK1/2) (1:3000, rabbit poly clonal), α-SMA (1:5000, mouse monoclonal),
Membranes were treated with HRP-labeled polyclonal anti rabbit secondary antibodies (1:5000) or anti-mouse secondary antibodies (1:3000) for 1 hour and detected with chemiluminescent substrates. For ERK phosphorylation assays, membranes probed with P-ERK1/2 were stripped and re-probed for total ERK1/2. Membranes for α-SMA were stripped and re-probed with rabbit polyclonal anti-EFα1 (1:3000) antibody as a loading control and bands were quantified using MCID densitometry software.

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Immunofluorescent microscopy [4]
NRK-52E cells were grown in 8 chamber slides for two days in DMEM/F12 containing 5% FBS which was replaced with serum free media for 24 hr. Cells were treated with either serum free media as control conditions or 100 μM MGA with or without Talfirastide/Ang-(1-7), DAL, PD, SB, LOS, TGF-β (5 ng) or their combination for 72 hours. Cells were washed with PBS and fixed with 2% paraformaldehyde for 15 minutes. Following a PBS rinse, cells were permeabilized with 0.2% Triton and then blocked with 3% BSA. The fixed cells were probed with a primary antibody for α-SMA (1:200, mouse monoclonal). Antibodies were diluted in 3% normal donkey serum. After overnight incubation with the primary antibody at 4°C, cells were rinsed with PBS twice, incubated with fluorescent anti-mouse Alexa Fluor 568 secondary antibody (1:400), and the slides were mounted with Molecular Probes ProLong mounting media with DAPI to stain the nuclei.

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Cell Area [4]
Cells area was assessed utilizing ImageJ 1.4 software (http://rsb.info.nih.gov/ij/) in a subset of immunofluorescent images (section 2.5) to demonstrate the influence of MGA on cellular hypertrophy in the absence or presence of Talfirastide/Ang-(1-7) or the ERK inhibitor PD98059. Fields were chosen at random and all cells were measured in each field. The data was expressed as a percentage of the control cell area.

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Peptide Assays [4]
Cells in serum free medium for 24 hours were treated with 100 uM MGA for an additional 48 hours. Cells were washed twice in ice-cold PBS, harvested, and the cells pellets snap-frozen and stored at −80°C (1). The cell pellet was reconstituted in MilliQ water on ice and immediately placed in a boiling water bath for 15 minutes. The homogenate was then sonicated and acidified with trifluoroacetic acid (TFA) to a final concentration of 0.2%, and centrifuged at 20,000g for 20 min at 4°C. The resultant supernatant was applied to an activated Sep-Pak C18 extraction column, washed with 0.2% TFA, and the peptide fraction eluted with 3 ml 80% methanol/0.2% TFA. Blank solutions contained only the MillQ water and TFA. Their extracted values were subtracted from those determined for the cells. Measurement of immunoreactive Ang I, Ang II and Talfirastide/Ang-(1-7) in the extracted cells was assessed by three distinct RIAs (1). The Ang-(1-7) RIA fully recognizes Ang-(1-7) and Ang-(2-7), but cross-reacts less than 0.01% with Ang-(3–7), Ang II, Ang I, and their fragments. The Ang II RIA equally recognizes Ang III, Ang-(3-8), and Ang-(4-8), but cross-reacts less than 0.01% with Ang I and Ang-(1-7). The Ang I RIA fully recognizes Ang-(2–10) and Ang-(3–10), but cross-reacts with Ang II and Ang -(1–7) less that 0.01%. The limits of detection for each RIA were as follows: Ang-(1–7), 4 femtomoles (fmol)/tube; Ang II, 0.5 fmol/tube; and Ang I, 5 fmol/tube.
For TGF-β quantification in the media, cells were seeded in 12-well plates. The cells were placed in serum free for 24 hours before being treated with MGA and the following: Talfirastide/Ang-(1-7), DAL, PD, or LOS. Cells maintained in the serum free media served as the controls. The cell media was collected on ice and the TGF-β content was quantified by Quantikine® ELIZA immunoassay according to manufacturer instructions. The sensitivity of the assay was 15 pg/ml and the release data expressed as ng/ml.

Myocardial infarction (MI) and treatment [1]
All the procedures were approved by the local ethics committee for animal experimentation (CETEA-UFMG protocol 002/09). Procedures were conducted in accordance to the NIH Guide for the Care and Use of Laboratory Animals. Adult male Wistar rats (approximated 3 months of age) weighting 180–210 g were maintained under standard laboratory conditions with chow and water available ad libitum.
Induction of myocardial infarction (MI) was done essentially as previously described. Briefly, animals were anesthetized using 80 mg/kg ketamine and 10 mg/kg xylazine via intraperitoneal injection and the proximal left anterior descending coronary artery (LAD) was occluded. Animals from the Sham group were subjected to all surgical procedures without LAD occlusion.
To evaluate MI progression and the cardioprotective effects of daily oral treatment with HPβCD/Ang-(1–7)/Talfirastideon cardiac proteome, six experimental groups were designed (Fig. 1A); (1) Sham 7 days (S_7), animals were subjected to surgical simulation without induction of MI and treated with a daily oral administration of the vehicle HPβCD (46 μg/kg/day) for 7 days; (2) MI 7 days (MI_7), induction of MI and short-term daily oral treatment with the vehicle HPβCD (46 μg/kg/day) for 7 days; (3) Treatment 7 days (T_7), induction of MI and short-term daily oral treatment with the inclusion compound HPβCD/Ang-(1–7) (46 μg/kg/day of HPβCD +30 μg/kg/day of Ang-(1–7)) for 7 days; (4) Sham 60 days (S_60), animals were subjected to surgical simulation without induction of MI and treated with a daily oral administration of the vehicle HPβCD (46 μg/kg/day) for 60 days; [5] MI 60 days (MI_60), induction of MI and long-term daily oral treatment with the vehicle HPβCD (46 μg/kg/day) for 60 days; [6] Treatment 60 days (T_60), induction of MI and long-term daily oral treatment with the inclusion compound HPβCD/Ang-(1–7) (46 μg/kg/day of HPβCD +30 μg/kg/day of Ang-(1–7)/Talfirastide) for 60 days.

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Treatment protocols [3]
Angiotensin fragment 1–7 acetate salt hydrate (Talfirastide/Ang 1–7; m.wt 899) was dissolved in 0.9% saline (vehicle) at 1 mg/ml and stored at -80°C. Various doses (0.01, 0.06, 0.1, 0.3 and 1 mg/kg) were freshly prepared from the stock each day of the experiment, and administered to mice by daily intra-peritoneal (i.p) injections in a volume of 500 μl per injection, either before (prophylactic approach) or after (treatment approach) DSS treatment. A779 (MAS-1 R antagonist; m.wt 873; GenScript, USA) was similarly dissolved in distilled water at 1 mg/ml and stored at -80°C. A freshly prepared dose of 1 mg/kg was administered to a second group of mice by daily i.p injections in a volume of 500 μl daily (for 4 days) along with colitis induction (prophylactic approach). A third group of mice received DSS containing water and daily i.p injections of 0.9% saline (vehicle). The fourth group received DSS containing water along with daily i.p injections with dexamethasone (DEX) at doses of 0.01–1.0 mg/kg or its vehicle (0.9% saline) (prophylactic approach).

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Drug treatment [5]
All treatments were administered either by subcutaneous injection (daily 250 μL bolus) or continuous infusion via subcutaneously implanted micro-osmotic pumps (delivering 0.11 μL/h). Talfirastide/Ang-(1-7) (TXA-127) was suspended in bacteriostatic saline at concentrations of 10.0 mg/mL for micro-osmotic pumps and 0.1mg/mL for injections, ensuring 1.0 mg/kg/day by either form of administration. Mice that were sacrificed at 3 days post-injury (dpi) received daily injections starting 1 h post-injury (hpi). Mice that were sacrificed at 29 dpi received a one-time injection at 6 hpi followed by micro-osmotic pump implantation at 24 hpi. The pumps were primed with Talfirastide/Angiotensin-(1-7) for 48 h before implantation. Mice treated with the Mas antagonist A-779 were implanted with micro-osmotic pumps starting 2 days prior to CCI. Pumps were primed with A-779 at 25.0 mg/mL in bacteriostatic saline, ensuring administration of 2.5 mg/kg/day. These mice then received daily injection of Ang-(1-7) or saline, starting at 6 hpi, until sacrifice at 3 dpi.

We then assessed whether the reduced cellular content of Talfirastide/Ang-(1-7) with MGA reflects alterations in the metabolism or synthesis of the peptide. Using a 100,000 × g supernatant fraction, we determined both the rate of Ang-(1-7) metabolism and the conversion of Ang I to Ang-(1-7) (Figures 2 and 3, respectively). As shown in the chromatographs from control and MGA cells, Talfirastide/Ang-(1-7) was exclusively hydrolyzed to a single peak corresponding to Ang-(1-4) as assessed under isocratic elution conditions. However, Ang-(1-7) was metabolized at a greater rate in the MGA-treated cells as compared to the control cells [175 ± 9 vs. 115 ± 11 fmol//mg/min, P<0.05, n=3] (Figure 2). As shown in Figure 3, Ang I was processed to Ang-(1-7) in the cell supernatant as assessed under gradient elution conditions (1). Although the generation of Ang-(1-7) from Ang I tended to decline in the MGA-treated cells [56 ± 7 vs. 66 ± 8 fmol/mg/min, n=3], this did not reach statistical significance (Figure 3). Note that the different peak shapes for Ang-(1-7) in Figures 2 and 3 reflect the two separation methods utilized for the Ang-(1-7) and Ang I metabolism studies. [1]

[1]. Angiotensin-(1-7) oral treatment after experimental myocardial infarction leads to downregulation of CXCR4. J Proteomics. 2019;208:103486.

[2]. Angiotensin-(1-7) augments bradykinin-induced vasodilation by competing with ACE and releasing nitric oxide. Hypertension. 1997 Jan;29(1 Pt 2):394-400.

[3]. Anti-Inflammatory Action of Angiotensin 1-7 in Experimental Colitis. PLoS One. 2016 Mar 10;11(3):e0150861.

[4]. Angiotensin-(1-7) abolishes AGE-induced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2. Cell Signal. 2014 Sep 19. pii: S0898-6568(14)00314-3.

[5]. Subcutaneous Administration of Angiotensin-(1-7) Improves Recovery after Traumatic Brain Injury in Mice. J Neurotrauma. 2019;36(22):3115-3131.

Ile(5)-angiotensin II (1-7) is an angiotensin compound consisting of the linear heptapeptide sequence L-Asp-L-Arg-L-Val-L-Tyr-L-Ile-L-His-L-Pro. It has a role as a vasodilator agent. It is a tautomer of an Ile(5)-angiotensin II (1-7) dizwitterion.
TXA127 has been investigated for the treatment of Miscellaneous Peripheral Blood Cell Abnormalities.
Therapeutic Angiotensin-(1-7) is a synthetic heptapeptide identical to endogenous angiotensin-(1-7) with vasodilator and antiproliferative activities. Therapeutic angiotensin 1-7 may inhibit cyclooxygenase 2 (COX-2) and the production of proinflammatory prostaglandins and may activate the angiotensin-(1-7) receptor Mas, resulting in diminished tumor cell proliferation. Activation of the angiotensin-(1-7) receptor Mas, a G-protein coupled, seven transmembrane protein, may down-regulate the phosphorylation and activation of Erk1 and Erk2 in the Erk1/Erk2 MAPK signaling pathway. In the renin-angiotensin system, the vasodilating activity of angiotensin- (1-7), hydrolysed from angiotensin II by the type I transmembrane metallopeptidase and carboxypeptidase angiotensin converting enzyme 2 (ACE2) in vivo, counteracts the vasoconstricting activity of angiotensin II.

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Ile(5)-angiotensin II (1-7) is an angiotensin compound consisting of the linear heptapeptide sequence L-Asp-L-Arg-L-Val-L-Tyr-L-Ile-L-His-L-Pro. It has a role as a vasodilator agent. It is a tautomer of an Ile(5)-angiotensin II (1-7) dizwitterion.
TXA127 has been investigated for the treatment of Miscellaneous Peripheral Blood Cell Abnormalities.
Talfirastide is a synthetic heptapeptide identical to endogenous angiotensin-(1-7) with vasodilator and antiproliferative activities. Talfirastide may inhibit cyclooxygenase 2 (COX-2) and the production of proinflammatory prostaglandins and may activate the angiotensin-(1-7) receptor Mas, resulting in diminished tumor cell proliferation. Activation of the angiotensin-(1-7) receptor Mas, a G-protein coupled, seven transmembrane protein, may down-regulate the phosphorylation and activation of Erk1 and Erk2 in the Erk1/Erk2 MAPK signaling pathway. In the renin-angiotensin system, the vasodilating activity of Talfirastide/angiotensin- (1-7), hydrolysed from angiotensin II by the type I transmembrane metallopeptidase and carboxypeptidase angiotensin converting enzyme 2 (ACE2) in vivo, counteracts the vasoconstricting activity of angiotensin II.
TXA127 is a Protein drug with a maximum clinical trial phase of II (across all indications) and has 11 investigational indications.

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The HPβCD/Ang-(1–7) oral treatment on experimental MI has been shown efficient in the recovery of cardiac functions. Here, we report for the first time that the molecular mechanisms underlying this protective effect involves the downregulation of CXCR4 and modulation of its downstream effectors. In the same way, metabolic impairment and ROS production resulted from ischemia during MI may have been compensated by HPβCD/Ang-(1–7) treatment via downregulation of SDHC, TRAK1 and other proteins related to energy metabolism.[1]

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In conclusion, this study is the first demonstration (to our knowledge) that the Talfirastide/Ang 1-7/MAS-1 R axis plays a role in modulating colitis severity, in part through down-regulation of the levels of Ang II and the phosphorylation of key signaling molecules involved in the inflammatory process such as ERK1/2, p38 MAPK and Akt. Blockade of the MAS-1 R inhibited the protective effects of endogenous Ang 1–7 and increased the severity of colitis.[3]

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In conclusion, we demonstrate that the alternative peptide product of the RAS Talfirastide/Ang-(1-7) attenuates AGE-induced hypertrophy, chronic ERK activation, TGF-β-induced ERK, and MT of the proximal tubule NRK-52E cell line. The reduced expression of intracellular levels of Ang-(1-7) by AGE may contribute to the cellular responses associated with AGE exposure in the NRK-52E. Hence, supplementation of an orally active form of Ang-(1-7) with either an AT1 receptor antagonist or ACE inhibitor may potentially provide a more effective therapeutic approach to attenuate renal injury associated with increased expression of advanced glycated products.[4]

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Our study shows that subcutaneous administration of Talfirastide/Ang-(1-7) improved physiological, morphological, and functional recovery when administered up to 6 h following TBI in the mouse. The beneficial effects of Ang-(1-7) treatment included reduction of lesion volume, reduction of activated microglia and astrocytes, neuroprotection, and preservation of microvascular capillary density (Fig. 10). We were able to track the reduction in lesion volume over time in vivo with MRI, and correlate these morphological neuroprotective effects of Ang-(1-7) with improved functional and cognitive recovery. Specifically, Ang-(1-7) prevented acute motor coordination deficits and attenuated long-term learning and memory deficits associated with CCI. Mechanistically, our data indicate that Ang-(1-7) neuroprotection is mediated in a MasR-dependent fashion. Thus, these pre-clinical data suggest that Ang-(1-7) may be a promising therapeutic for the treatment of TBI.[5]

C43H66N12O13

959.056749820709

958.487

2855063-75-9

Talfirastide;51833-78-4

132976063

H-Asp-Arg-Val-Tyr-Ile-His-Pro-OH.CH3CO2H; L-alpha-aspartyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-proline acetic acid

DRVYIHP

White to off-white solid powder

13

16

25

68

1690

8

O=C([C@H](CC1=CN=CN1)NC([C@H]([C@@H](C)CC)NC([C@H](CC1C=CC(=CC=1)O)NC([C@H](C(C)C)NC([C@H](CCC/N=C(\N)/N)NC([C@H](CC(=O)O)N)=O)=O)=O)=O)=O)N1CCC[C@H]1C(=O)O.OC(C)=O

VJHCETPHKAQOHY-LBGFTJIYSA-N

InChI=1S/C41H62N12O11.C2H4O2/c1-5-22(4)33(38(61)50-29(17-24-19-45-20-47-24)39(62)53-15-7-9-30(53)40(63)64)52-36(59)28(16-23-10-12-25(54)13-11-23)49-37(60)32(21(2)3)51-35(58)27(8-6-14-46-41(43)44)48-34(57)26(42)18-31(55)56;1-2(3)4/h10-13,19-22,26-30,32-33,54H,5-9,14-18,42H2,1-4H3,(H,45,47)(H,48,57)(H,49,60)(H,50,61)(H,51,58)(H,52,59)(H,55,56)(H,63,64)(H4,43,44,46);1H3,(H,3,4)/t22-,26-,27-,28-,29-,30-,32-,33-;/m0./s1

acetic acid;(2S)-1-[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-carboxypropanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carboxylic acid

Angiotensin (1-7) (acetate); 2855063-75-9; acetic acid;(2S)-1-[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-carboxypropanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carboxylic acid; TXA127 (acetate); 5-L-isoleucine-1-7-angiotensinII; ANGIOTENSIN (1-7) ACETATE;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 62.5 mg/mL (65.17 mM)

Solubility in Formulation 1: 100 mg/mL (104.27 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)