Galanin 2 Receptor: A Novel Target for a Subset of Pancreatic Ductal Adenocarcinoma.

Galanin is a 30 amino acid peptide that stimulates three subtype receptors (GAL1-3R). M89b is a lanthionine-stabilized, C-terminally truncated galanin analog that specifically stimulates GAL2R. We investigated the potential of M89b as a therapeutic for pancreatic ductal adenocarcinoma (PDAC) and assessed its safety. The anti-tumor activity of subcutaneously injected M89b on the growth of patient-derived xenografts of PDAC (PDAC-PDX) in mice was investigated. In addition, the safety of M89b was assessed in vitro using a multi-target panel to measure the off-target binding and modulation of enzyme activities. In a PDAC-PDX with a high GAL2R expression, M89b completely inhibited the growth of the tumor (p < 0.001), while in two PDAC-PDXs with low GAL2R expression, low or negligeable inhibition of tumor growth was measured, and in the PDX without GAL2R expression no influence on the tumor growth was observed. The M89b treatment of the GAL2R high-PDAC-PDX-bearing mice led to a reduction in the expression of RacGap1 (p < 0.05), PCNA (p < 0.01), and MMP13 (p < 0.05). In vitro studies involving a multi-target panel of pharmacologically relevant targets revealedexcellent safety of M89b. Our data indicated that GAL2R is a safe and valuable target for treating PDACs with high GAL2R expression.

An Engineered Double Lipid II Binding Motifs-Containing Lantibiotic Displays Potent and Selective Antimicrobial Activity against Enterococcus faecium.

Lipid II is an essential precursor for bacterial cell wall biosynthesis and thereby an important target for various antibiotics. Several lanthionine-containing peptide antibiotics target lipid II with lanthionine-stabilized lipid II binding motifs. Here, we used the biosynthesis system of the lantibiotic nisin to synthesize a two-lipid II binding motifs-containing lantibiotic, termed TL19, which contains the N-terminal lipid II binding motif of nisin and the distinct C-terminal lipid II binding motif of one peptide of the two-component haloduracin (i.e., HalA1). Further characterization demonstrated that (i) TL19 exerts 64-fold stronger antimicrobial activity against Enterococcus faecium than nisin(1-22), which has only one lipid II binding site, and (ii) both the N- and C-terminal domains are essential for the potent antimicrobial activity of TL19, as evidenced by mutagenesis of each single and the double domains. These results show the feasibility of a new approach to synthesize potent lantibiotics with two different lipid II binding motifs to treat specific antibiotic-resistant pathogens.

Does activation of the protective Renin-Angiotensin System have therapeutic potential in COVID-19?

Infection of lung cells by the corona virus results in a loss of the balance between, on the one hand, angiotensin II-mediated stimulation of the angiotensin II type 1 receptor and, on the other hand, stimulation of the angiotensin II type 2 receptor and/or the Mas receptor. The unbalanced enhanced stimulation of the angiotensin II type 1 receptor causes inflammation, edema and contributes to the pathogenesis of severe acute respiratory distress syndrome. Here we hypothesize that stable, receptor-specific agonists of the angiotensin II type 2 receptor and of the Mas receptor are molecular medicines to treat COVID-19 patients. These agonists have therapeutic potential in the acute disease but in addition may reduce COVID-19-associated long-term pulmonary dysfunction and overall end-organ damage of this disease.

Biosynthesis of lanthionine-constrained agonists of G protein-coupled receptors.

The conformation with which natural agonistic peptides interact with G protein-coupled receptor(s) (GPCR(s)) partly results from intramolecular interactions such as hydrogen bridges or is induced by ligand-receptor interactions. The conformational freedom of a peptide can be constrained by intramolecular cross-links. Conformational constraints enhance the receptor specificity, may lead to biased activity and confer proteolytic resistance to peptidic GPCR agonists. Chemical synthesis allows to introduce a variety of cross-links into a peptide and is suitable for bulk production of relatively simple lead peptides. Lanthionines are thioether bridged alanines of which the two alanines can be introduced at different distances in chosen positions in a peptide. Thioether bridges are much more stable than disulfide bridges. Biosynthesis of lanthionine-constrained peptides exploiting engineered Gram-positive or Gram-negative bacteria that contain lanthionine-introducing enzymes constitutes a convenient method for discovery of lanthionine-stabilized GPCR agonists. The presence of an N-terminal leader peptide enables dehydratases to dehydrate serines and threonines in the peptide of interest after which a cyclase can couple the formed dehydroamino acids to cysteines forming (methyl)lanthionines. The leader peptide also guides the export of the formed lanthionine-containing precursor peptide out of Gram-positive bacteria via a lanthipeptide transporter. An engineered cleavage site in the C-terminus of the leader peptide allows to cleave off the leader peptide yielding the modified peptide of interest. Lanthipeptide GPCR agonists are an emerging class of therapeutics of which a few examples have demonstrated high efficacy in animal models of a variety of diseases. One lanthipeptide GPCR agonist has successfully passed clinical Phase Ia.

Genetically Encoded Libraries of Constrained Peptides.

Many therapeutic peptides can still be improved with respect to target specificity, target affinity, resistance to peptidases/proteases, physical stability, and capacity to pass through membranes required for oral delivery. Several modifications can improve the peptides' properties, in particular those that impose (a) conformational constraint(s). Screening of constrained peptides and the identification of hits is greatly facilitated by the generation of genetically encoded libraries. Recent breakthrough bacterial, phage, and yeast display screening systems of ribosomally synthesized post-translationally constrained peptides, particularly those of lanthipeptides, are earning special attention. Here we provide an overview of display systems for constrained, genetically encoded peptides and indicate prospects of constrained peptide-displaying phage and bacterial systems as such in vivo.

Mutagenesis of nisin's leader peptide proline strongly modulates export of precursor nisin.

The lantibiotic nisin is produced by Lactococcus lactis as a precursor peptide comprising a 23 amino acid leader peptide and a 34 amino acid post-translationally modifiable core peptide. We previously demonstrated that the conserved FNLD part of the leader is essential for intracellular enzyme-catalyzed introduction of lanthionines in the core peptide and also for transporter-mediated export, whereas other positions are subject to large mutational freedom. We here demonstrate that, in the absence of the extracellular leader peptidase, NisP, export of precursor nisin via the modification and transporter enzymes, NisBTC, is strongly affected by multiple substitutions of the leader residue at position -2, but not by substitution of positions in the vicinity of this site. Export levels of precursor nisin increased by more than 70% for position -2 mutants Asp, Thr, Ser, Trp, Lys, Val and decreased more than 70% for Cys, His, Met. In a strain with leader peptidase, the Pro-2Lys and Pro-2Asp precursor nisins were less efficiently cleaved by NisP than wild type precursor nisin. Taken together, the wild type precursor nisin with a proline at position -2 allows balanced export and cleavage efficiencies by precursor nisin's transporter and leader peptidase.

Agonists of galanin subtype 2 receptor may prevent pancreatic cancer and agonists of angiotensin II type 2 receptor may prevent colorectal cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a dreadful disease with poor prognosis. While the prognosis of colorectal carcinoma (CRC) is better than that of PDAC, it still is the second-leading cause of cancer deaths worldwide. Recently, a (methyl)lanthionine-stabilized, highly receptor-specific agonist of galanin subtype 2 (GAL2) receptor inhibited the growth of GAL2 receptor-expressing patient-derived xenografts (PDX) of pancreatic cancer. Furthermore, a lanthionine-constrained agonist of angiotensin II type 2 (AT2) receptor inhibited PDX of colorectal cancer in mice. Stimulation of GAL2 receptor may modulate immune surveillance and inhibits PDAC via cell cycle inhibition and apoptosis. Consistent with GAL2 receptor-mediated tumor inhibition, for PDAC, survival is much higher for patients with high GAL2 receptor expression. Importantly, a (methyl)lanthionine-stabilized GAL2 receptor-specific agonist enhances expression of GAL2 receptor, not only in PDAC-PDX but also in healthy tissue indicating therapeutic and preventive potentials for GAL2 receptor agonists. AT2 receptor is interacting with four tumor suppressor proteins, Src homology phosphatase 1, Src homology phosphatase 2, Promyelocytic Leukemia Zinc Finger protein and Microtuble-Associated Scaffold Protein1, the latter also known as Angiotensin-II type 2 receptor-Interacting Protein. Pathways linked to these tumor suppressor proteins may enhance immune surveillance, prevent carcinogenesis, counter proliferation and stimulate apoptosis. Taken together, current data are prompting the hypothesis of a prophylactic treatment option with stable, specific and safe agonists of GAL2 receptor and AT2 receptor to prevent the emergence of pancreatic and colorectal cancer in individuals at risk.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Evolving views on the first two ligands of the angiotensin II type 2 receptor. From putative antagonists to potential agonists?

The renin-angiotensin system is one of the most complex regulatory systems that controls multiple organ functions. One of its key components, angiotensin II (Ang II), stimulates two G-protein coupled class A receptors: the Ang II type 1 (AT1) receptor and the Ang II type 2 (AT2) receptor. While stimulation of the AT1 receptor causes G-protein-dependent signaling and arrestin recruitment, the AT2 receptor seems to have a constitutively active-like conformation and appears to act via G-protein-dependent and -independent pathways. Overstimulation of the AT1 receptor may lead to unwanted effects like inflammation and fibrosis. In contrast, stimulation of the AT2 receptor leads to opposite effects thus restoring the balance. However, the role of the AT2 receptor has become controversial due to beneficial effects of putative AT2 receptor antagonists. The two first synthetic AT2 receptor-selective ligands, peptide CGP42112 and small molecule PD123319, were initially both considered antagonists. CGP42112 was subsequently considered a partial agonist and it was recently demonstrated to be a full agonist. Based on the search-term PD123319 in Pubmed, 1652 studies have investigated putative AT2 receptor antagonist PD123319. Here, we put forward literature that shows beneficial effects of PD123319 alone, even at doses too low for antagonist efficacy. These beneficial effects appear compatible with agonist-like activity via the AT2 receptor. Taken together, a more consistent image of a therapeutic role of stimulated AT2 receptor emerges which may clarify current controversies.

AT2 receptor agonist LP2 restores respiratory function in a rat model of bleomycin-induced lung remodelling.

This study aimed to evaluate the prophylactic and therapeutic potential of angiotensin II type 2 receptor peptide agonist LP2 in bleomycin-induced airway and cardiac remodeling in rats. Male Wistar rats were intratracheally instillated with bleomycin. Animals of a prophylactic arm received LP2 from day 0 at intraperitoneal doses of 1, 3 or 10 µg/kg/d, whereas animals from a therapeutic arm received this LP2 treatment from day 7. On day 28 direct lung mechanics were determined and cardiac and lung tissues were collected and (histo)morphologically assessed. Prophylactic LP2 at 1 µg/kg/d with bleomycin, versus bleomycin alone, significantly improved the airway pressure responses at fixed inflation of 4 ml (p < 0.05) and 7 ml volume (p < 0.05), static compliance (p < 0.01), inspiratory capacity (p < 0.05), lung tolerance of increased volume (p < 0.0001), right to left ventricular hypertrophy (p < 0.05). Therapeutic regime showed a similar trend as the prophylactic arm but was less effective, mostly lacking significance. However, and importantly, therapeutic LP2 at 1 µg/kg/d significantly decreased mRNA expression of collagen 1A1 (p < 0.01), of Connective Tissue Growth Factor 1 (p < 0.05) and of Tissue MetalloPeptidase inhibitor 1 (p < 0.05). In conclusion, a very low dose of 1 µg/kg/d LP2 has capacity to counter bleomycin-induced impairment of lung functioning and consequent cardiac remodeling.

Substrate recognition and specificity of the NisB protein, the lantibiotic dehydratase involved in nisin biosynthesis.

Nisin is a posttranslationally modified antimicrobial peptide containing the cyclic thioether amino acids lanthionine and methyllanthionine. Although much is known about its antimicrobial activity and mode of action, knowledge about the nisin modification process is still rather limited. The dehydratase NisB is believed to be the initial interaction partner in modification. NisB dehydrates specific serine and threonine residues in prenisin, whereas the cyclase NisC catalyzes the (methyl)lanthionine formation. The fully modified prenisin is exported and the leader peptide is cleaved off by the extracellular protease NisP. Light scattering analysis demonstrated that purified NisB is a dimer in solution. Using size exclusion chromatography and surface plasmon resonance, the interaction of NisB and prenisin, including several of its modified derivatives, was studied. Unmodified prenisin binds to NisB with an affinity of 1.05 ± 0.25 µm, whereas the dehydrated and the fully modified derivatives bind with respective affinities of 0.31 ± 0.07 and 10.5 ± 1.7 µm. The much lower affinity for the fully modified prenisin was related to a >20-fold higher off-rate. For all three peptides the stoichiometry of binding was 1:1. Active nisin, which is the equivalent of fully modified prenisin lacking the leader peptide did not bind to NisB, nor did prenisin in which the highly conserved FNLD box within the leader peptide was mutated to AAAA. Taken together our data indicate that the leader peptide is essential for initial recognition and binding of prenisin to NisB.

Determining sites of interaction between prenisin and its modification enzymes NisB and NisC.

Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L. lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB Δ(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC.

Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1-7) or an angiotensin II receptor antagonist.

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome. Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin (Ang) II have been implicated in the pathogenesis of ARDS. A counter-regulatory enzyme of ACE, ie ACE2 that degrades Ang II to Ang-(1-7), offers a promising novel treatment modality for this syndrome. As the involvement of ACE and ACE2 in ARDS is still unclear, this study investigated the role of these two enzymes in an animal model of ARDS. ARDS was induced in rats by intratracheal administration of LPS followed by mechanical ventilation. During ventilation, animals were treated with saline (placebo), losartan (Ang II receptor antagonist), or with a protease-resistant, cyclic form of Ang-(1-7) [cAng-(1-7)]. In bronchoalveolar lavage fluid (BALF) of ventilated LPS-exposed animals, ACE activity was enhanced, whereas ACE2 activity was reduced. This was matched by enhanced BALF levels of Ang II and reduced levels of Ang-(1-7). Therapeutic intervention with cAng-(1-7) attenuated the inflammatory mediator response, markedly decreased lung injury scores, and improved lung function, as evidenced by increased oxygenation. These data indicate that ARDS develops, in part, due to reduced pulmonary levels of Ang-(1-7) and that repletion of this peptide halts the development of ARDS.

Bacterial display and screening of posttranslationally thioether-stabilized peptides.

A major hurdle in the application of therapeutic peptides is their rapid degradation by peptidases. Thioether bridges effectively protect therapeutic peptides against breakdown, thereby strongly increasing bioavailability, enabling oral and pulmonary delivery and potentially significantly optimizing the receptor interaction of selected variants. To efficiently select optimal variants, a library of DNA-coupled thioether-bridged peptides is highly desirable. Here, we present a unique cell surface display system of thioether-bridged peptides and successfully demonstrate highly selective screening. Peptides are posttranslationally modified by thioether bridge-installing enzymes in Lactococcus lactis, followed by export and sortase-mediated covalent coupling to the lactococcal cell wall. This allows the combinatorial optimization and selection of medically and economically highly important therapeutic peptides with strongly enhanced therapeutic potential.

Requirements of the engineered leader peptide of nisin for inducing modification, export, and cleavage.

Nisin A is a pentacyclic peptide antibiotic produced by Lactococcus lactis. The leader peptide of prenisin keeps nisin inactive and has a role in inducing NisB- and NisC-catalyzed modifications of the propeptide and NisT-mediated export. The highly specific NisP cleaves off the leader peptide from fully modified and exported prenisin. We present here a detailed mutagenesis analysis of the nisin leader peptide. For alternative cleavage, we successfully introduced a putative NisP autocleavage site and sites for thrombin, enterokinase, Glu-C, and factor Xa in the C-terminal part of the leader peptide. Replacing residue F-18 with Trp or Thr strongly reduced production. On the other hand, D-19A, F-18H, F-18M, L-16D, L-16K, and L-16A enhanced production. Substitutions within and outside the FNLD box enhanced or reduced the transport efficiency. None of the above substitutions nor even an internal 6His tag from positions -13 to -8 had any effect on the capacity of the leader peptide to induce NisB and NisC modifications. Therefore, these data demonstrate a large mutational freedom. However, simultaneous replacement of the FNLD amino acids by four alanines strongly reduced export and even led to a complete loss of the capacity to induce modifications. Reducing the leader peptide to MSTKDFNLDLR led to 3- or 4-fold dehydration. Taken together, the FNLD box is crucial for inducing posttranslational modifications.

LP2, the first lanthipeptide GPCR agonist in a human pharmacokinetics and safety study.

Introduction of a lanthionine into a peptide may enhance target affinity, target specificity and proteolytic resistance. This manuscript reports preclinical safety studies and the first-in-human study with the lanthipeptide AT2R agonist LP2, a structural analog of cAng-(1-7), whose N-terminus was protected against aminopeptidases by the presence of a d-lysine. None of the preclinical studies, including an in vitro multitarget panel, behavioral, respiratory and cardiovascular measurements, genotoxicity and toxicity studies in rat and dog, posed any safety concern. Due to lack of toxicity the maximum tolerated dose was not reached neither in rat nor in dog. In the human dose escalation study, healthy male volunteers received a single 1 mL subcutaneous injection (0.001 mg, 0.01 mg or 0.1 mg) of LP2 or matching placebo. In contrast to angiotensin II which has a T1/2 in plasma of < 1 min, LP2 has a T1/2 of approximately 2.1-2.6 hours. The fraction of the dose excreted unchanged in urine ranged from 84.73 ± 10.4 % at a dose of 0.001 mg to 66.4 ± 3.9 % at 0.1 mg. There were no deaths, serious adverse events or subject withdrawals as a result of an adverse event. The incidence of adverse events was 16.7 %; each was mild in severity. One adverse event, peripheral coldness, was considered to be possibly related to LP2 at 0.001 mg LP2. None of the results was considered to pose a clinically relevant safety concern. This study supports the potential for the therapeutic use of lanthipeptides.

Intranasal Delivery of a Methyllanthionine-Stabilized Galanin Receptor-2-Selective Agonist Reduces Acute Food Intake.

The regulatory (neuro)peptide galanin is widely distributed in the central and peripheral nervous systems, where it mediates its effects via three G protein-coupled receptors (GAL1-3R). Galanin has a vast diversity of biological functions, including modulation of feeding behavior. However, the clinical application of natural galanin is not practicable due to its rapid in vivo breakdown by peptidases and lack of receptor subtype specificity. Much effort has been put into the development of receptor-selective agonists and antagonists, and while receptor selectivity has been attained to some degree, most ligands show overlapping affinity. Therefore, we aimed to develop a novel ligand with specificity to a single galanin receptor subtype and increased stability. To achieve this, a lanthionine amino acid was enzymatically introduced into a galanin-related peptide. The residue's subsequent cyclization created a conformational constraint which increased the peptide's receptor specificity and proteolytic resistance. Further exchange of certain other amino acids resulted in a novel methyllanthionine-stabilized galanin receptor agonist, a G1pE-T3N-S6A-G12A-methyllanthionine[13-16]-galanin-(1-17) variant, termed M89b. M89b has exclusive specificity for GAL2R and a prolonged half-life in serum. Intranasal application of M89b to unfasted rats significantly reduced acute 24 h food intake inducing a drop in body weight. Combined administration of M89b and M871, a selective GAL2R antagonist, abolished the anorexigenic effect of M89b, indicating that the effect of M89b on food intake is indeed mediated by GAL2R. This is the first demonstration of in vivo activity of an intranasally administered lanthipeptide. Consequently, M89b is a promising candidate for clinical application as a galanin-related peptide-based therapeutic.

Production of a class II two-component lantibiotic of Streptococcus pneumoniae using the class I nisin synthetic machinery and leader sequence.

Recent studies showed that the nisin modification machinery can successfully dehydrate serines and threonines and introduce lanthionine rings in small peptides that are fused to the nisin leader sequence. This opens up exciting possibilities to produce and engineer larger antimicrobial peptides in vivo. Here we demonstrate the exploitation of the class I nisin production machinery to generate, modify, and secrete biologically active, previously not-yet-isolated and -characterized class II two-component lantibiotics that have no sequence homology to nisin. The nisin synthesis machinery, composed of the modification enzymes NisB and NisC and the transporter NisT, was used to modify and secrete a putative two-component lantibiotic of Streptococcus pneumoniae. This was achieved by genetically fusing the propeptide-encoding sequences of the spr1765 (pneA1) and spr1766 (pneA2) genes to the nisin leader-encoding sequence. The chimeric prepeptides were secreted out of Lactococcus lactis, purified by cation exchange fast protein liquid chromatography, and further characterized. Mass spectrometry analyses demonstrated the presence and partial localization of multiple dehydrated serines and/or threonines and (methyl)lanthionines in both peptides. Moreover, after cleavage of the leader peptide from the prepeptides, both modified propeptides displayed antimicrobial activity against Micrococcus flavus. These results demonstrate that the nisin synthetase machinery can be successfully used to modify and produce otherwise difficult to obtain antimicrobially active lantibiotics.

Microbial engineering of dehydro-amino acids and lanthionines in non-lantibiotic peptides.

This minireview focuses on the use of bacteria to introduce dehydroresidues and (methyl)lanthionines in (poly)peptides. It mainly describes the broad exploitation of bacteria containing lantibiotic enzymes for the engineering of these residues in a wide variety of peptides in particular in peptides unrelated to lantibiotics. Lantibiotic dehydratases dehydrate serines and threonines present in peptides preceded by a lantibiotic leader peptide thus forming dehydroalanine and dehydrobutyrine, respectively. These dehydroresidues can be coupled to cysteines thus forming (methyl)lanthionines. This coupling is catalysed by lantibiotic cyclases. The design, synthesis, and export of microbially engineered dehydroresidue and or lanthionine-containing peptides in non-lantibiotic peptides are reviewed, illustrated by some examples which demonstrate the high relevance of these special residues. This minireview is the first with special focus on the microbial engineering of nonlantibiotic peptides by exploiting lantibiotic enzymes.

Cyclic angiotensin-(1-7) contributes to rehabilitation of animal performance in a rat model of cerebral stroke.

Peptidase-resistant, lanthionine-stabilized angiotensin-(1-7), termed cAng-(1-7), has shown therapeutic efficacy in animal models of cardiovascular, metabolic, kidney and pulmonary disease. Goal of the present study was testing the capacity of subcutaneously administered cAng-(1-7) to induce rehabilitation of animal performance in the transient middle cerebral artery occlusion rat model of cerebral stroke. 24 h after ischemic stroke induction, cAng-(1-7) was administered for 28 days at a dose of 500 µg/kg/day, either daily via subcutaneous injection or continuously via an alzet pump. Both ways of administration of cAng-(1-7) were equally effective. Measurements were continued until day 50. Compared to vehicle, cAng-(1-7) clearly demonstrated significantly increased capillary density (p < 0.01) in the affected hemisphere and improved motor and somatosensory functioning. The modified neurological severity score (p < 0.001 at days 15 and 50), stepping test (p < 0.001 at days 36-50), forelimb placement test (p < 0.001 at day 50), body swing test (p < 0.001 at days 43 and 50) all demonstrated that cAng-(1-7) caused significantly improved animal performance. Taken together the data convincingly indicate rehabilitating capacity of subcutaneously injected cAng-(1-7) in cerebral ischemic stroke.