Ac-SDKP attenuates ER stress-stimulated collagen production in cardiac fibroblasts by inhibiting CHOP-mediated NF-κB expression.

Inflammation and cardiac fibrosis are prevalent pathophysiologic conditions associated with hypertension, cardiac remodeling, and heart failure. Endoplasmic reticulum (ER) stress triggers the cells to activate unfolded protein responses (UPRs) and upregulate the ER stress chaperon, enzymes, and downstream transcription factors to restore normal ER function. The mechanisms that link ER stress-induced UPRs upregulation and NF-κB activation that results in cardiac inflammation and collagen production remain elusive. N-Acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a natural tetrapeptide that negatively regulates inflammation and fibrosis, has been reported. Whether it can inhibit ER stress-induced collagen production in cardiac fibroblasts remains unclear. Thus, we hypothesized that Ac-SDKP attenuates ER stress-stimulated collagen production in cardiac fibroblasts by inhibiting CHOP-mediated NF-κB expression. We aimed to study whether Ac-SDKP inhibits tunicamycin (TM)-induced ER stress signaling, NF-κB signaling, the release of inflammatory cytokine interleukin-6, and collagen production in human cardiac fibroblasts (HCFs). HCFs were pre-treated with Ac-SDKP (10 nM) and then stimulated with TM (0.25 µg/mL). We found that Ac-SDKP inhibits TM-induced collagen production by attenuating ER stress-induced UPRs upregulation and CHOP/NF-κB transcriptional signaling pathways. CHOP deletion by specific shRNA maintains the inhibitory effect of Ac-SDKP on NF-κB and type-1 collagen (Col-1) expression at both protein and mRNA levels. Attenuating ER stress-induced UPR sensor signaling by Ac-SDKP seems a promising therapeutic strategy to combat detrimental cardiac inflammation and fibrosis.

Abnormal Expression of Prolyl Oligopeptidase (POP) and Its Catalytic Products Ac-SDKP Contributes to the Ovarian Fibrosis Change in Polycystic Ovary Syndrome (PCOS) Mice.

Polycystic ovary syndrome (PCOS) is an endocrine disorder and metabolic syndrome. Ovarian fibrosis pathological change in PCOS has gradually attracted people's attention. In this study, we constructed a PCOS mouse model through the use of dehydroepiandrosterone. Sirius red staining showed that the ovarian tissues in PCOS mice had obvious fibrosis. Prolyl oligopeptidase (POP) is a serine protease and N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is its catalytic product. Studies show that abnormal expression and activity of POP and Ac-SDKP are closely related to tissue fibrosis. It was found that the expression of POP and Ac-SDKP was decreased in the ovaries of PCOS mice. Further studies showed that POP and Ac-SDKP promoted the expression of matrix metalloproteinases 2 (MMP-2) expression and decreased the expression of transforming growth factor beta 1 (TGF-ß1) in granulosa cells. Hyperandrogenemia is a typical symptom of PCOS. We found that testosterone induced the low expression of POP and MMP2 and high expression of TGF-ß1 in granulosa cells. POP overexpression and Ac-SDKP treatment inhibited the effect of testosterone on TGF-ß1 and MMP2 in vitro and inhibited ovarian fibrosis in the PCOS mouse model. In conclusion, PCOS ovarian tissue showed obvious fibrosis. Low expression of POP and Ac-SDKP and changes in fibrotic factors contribute to the ovarian pathological fibrosis induced by androgen.

Phospholipid Encapsulation of an Anti-Fibrotic Endopeptide to Enhance Cellular Uptake and Myocardial Retention.

BACKGROUND: Cardioprotective effects of N-acetyl-ser-asp-lys-pro (Ac-SDKP) have been reported in preclinical models of myocardial remodeling. However, the rapid degradation of this endogenous peptide in vivo limits its clinical use. METHOD: To prolong its bioavailability, Ac-SDKP was encapsulated by phosphocholine lipid bilayers (liposomes) similar to mammalian cell membranes. The physical properties of the liposome structures were assessed by dynamic light scattering and scanning electron microscopy. The uptake of Ac-SDKP by RAW 264.7 macrophages and human and murine primary cardiac fibroblasts was confirmed by fluorescence microscopy and flow cytometry. Spectrum computerized tomography and competitive enzyme-linked immunoassays were performed to measure the ex vivo cardiac biodistribution of Ac-SDKP. The biological effects of this novel synthetic compound were examined in cultured macrophages and cardiac fibroblasts and in a murine model of acute myocardial infarction induced by permanent coronary artery ligation. RESULTS: A liposome formulation resulted in the greater uptake of Ac-SDKP than the naked peptide by cultured RAW 264.7 macrophages and cardiac fibroblasts. Liposome-delivered Ac-SDKP decreased fibroinflammatory genes in cultured cardiac fibroblasts co-treated with TGF-ß1 and macrophages stimulated with LPS. Serial tissue and serum immunoassays showed the high bioavailability of Ac-SDKP in mouse myocardium and in circulation. Liposome-delivered Ac-SDKP improved cardiac function and reduced myocardial fibroinflammatory responses in mice with acute myocardial infarction. CONCLUSION: Encapsulation of Ac-SDKP in a cell membrane-like phospholipid bilayer enhances its plasma and tissue bioavailability and offers cardioprotection against ischemic myocardial injury. Future clinical trials can use this novel approach to test small protective endogenous peptides in myocardial remodeling.

[Review on biological effects and mechanism of Ac-SDKP].

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is an endogenous short peptide produced through the continuous hydrolysis of Thymosin ß4 by meprin-α and prolyl oligopeptidase. It has the functions of immune regulation, promoting angiogenesis, tumorigenesis and anti-fibrosis in organs. In this paper, according to some our research results and related literatures in recent years, a review of Ac-SDKP research progress was written.

Knockout of ACE-N facilitates improved cardiac function after myocardial infarction.

Angiotensin-converting enzyme (ACE) hydrolyzes N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) into inactive fragments through its N-terminal site (ACE-N). We previously showed that Ac-SDKP mediates ACE inhibitors' cardiac effects. Whether increased bioavailability of endogenous Ac-SDKP caused by knocking out ACE-N also improves cardiac function in myocardial infarction (MI)-induced heart failure (HF) is unknown. Wild-type (WT) and ACE-N knockout (ACE-NKO) mice were subjected to MI by ligating the left anterior descending artery and treated with vehicle or Ac-SDKP (1.6 mg/kg/day, s.c.) for 5 weeks, after which echocardiography was performed and left ventricles (LV) were harvested for histology and molecular biology studies. ACE-NKO mice showed increased plasma Ac-SDKP concentrations in both sham and MI group compared to WT. Exogenous Ac-SDKP further increased its circulating concentrations in WT and ACE-NKO. Shortening (SF) and ejection (EF) fractions were significantly decreased in both WT and ACE-NKO mice post-MI, but ACE-NKO mice exhibited significantly lesser decrease. Exogenous Ac-SDKP ameliorated cardiac function post-MI only in WT but failed to show any additive improvement in ACE-NKO mice. Sarcoendoplasmic reticulum calcium transport ATPase (SERCA2), a marker of cardiac function and calcium homeostasis, was significantly decreased in WT post-MI but rescued with Ac-SDKP, whereas ACE-NKO mice displayed less loss of SERCA2 expression. Our study demonstrates that gene deletion of ACE-N resulted in improved LV cardiac function in mice post-MI, which is likely mediated by increased circulating Ac-SDKP and minimally reduced expression of SERCA2. Thus, future development of specific and selective inhibitors for ACE-N could represent a novel approach to increase endogenous Ac-SDKP toward protecting the heart from post-MI remodeling.

Thymosin ß4 and the anti-fibrotic switch

Wound healing involves a rapid response to the injury by circulating cells, followed by inflammation with an influx of inflammatory cells that release various factors. Soon after, cellular proliferation begins to replace the damaged cells and extracellular matrix, and then tissue remodeling restores normal tissue function. Various factors can lead to pathological wound healing when excessive and irreversible connective tissue/extracellular matrix deposition occurs, resulting in fibrosis. The process is initiated when immune cells, such as macrophages, release soluble factors that stimulate fibroblasts. TGFß is the most well-characterized macrophage derived pro-fibrotic mediator. Other soluble mediators of fibrosis include connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), and interleukin 10 (IL-10). Thymosin ß4 (Tß4) has shown therapeutic benefit in preventing fibrosis/scarring in various animal models of fibrosis/scarring. The mechanism of action of Tß4 appears related, in part, to a reduction in the inflammatory response, including a reduction in macrophage infiltration, decreased levels of TGFß and IL-10, and reduced CTGF activation, resulting in both prevention of fibroblast conversion to myofibroblasts and production of normally aligned collagen fibers. The amino N-terminal end of Tß4, SDKP (serine-aspartate-lysine-proline), appears to contain the majority of anti-fibrotic activity and has shown excellent efficacy in many animal models of fibrosis, including liver, lung, heart, and kidney fibrosis. Ac-SDKP not only prevents fibrosis but can reverse fibrosis. Unanswered questions and future directions will be presented with regard to therapeutic uses alone and in combination with already approved drugs for fibrosis.

Review on biological effects and mechanism of Ac-SDKP / 中华劳动卫生职业病杂志

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is an endogenous short peptide produced through the continuous hydrolysis of Thymosin β4 by meprin-α and prolyl oligopeptidase. It has the functions of immune regulation, promoting angiogenesis, tumorigenesis and anti-fibrosis in organs. In this paper, according to some our research results and related literatures in recent years, a review of Ac-SDKP research progress was written.

The Role of Tß4-POP-Ac-SDKP Axis in Organ Fibrosis.

Fibrosis is a pathological process in which parenchymal cells are necrotic and excess extracellular matrix (ECM) is accumulated due to dysregulation of tissue injury repair. Thymosin ß4 (Tß4) is a 43 amino acid multifunctional polypeptide that is involved in wound healing. Prolyl oligopeptidase (POP) is the main enzyme that hydrolyzes Tß4 to produce its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) which is found to play a role in the regulation of fibrosis. Accumulating evidence suggests that the Tß4-POP-Ac-SDKP axis widely exists in various tissues and organs including the liver, kidney, heart, and lung, and participates in the process of fibrogenesis. Herein, we aim to elucidate the role of Tß4-POP-Ac-SDKP axis in hepatic fibrosis, renal fibrosis, cardiac fibrosis, and pulmonary fibrosis, as well as the underlying mechanisms. Based on this, we attempted to provide novel therapeutic strategies for the regulation of tissue damage repair and anti-fibrosis therapy. The Tß4-POP-Ac-SDKP axis exerts protective effects against organ fibrosis. It is promising that appropriate dosing regimens that rely on this axis could serve as a new therapeutic strategy for alleviating organ fibrosis in the early and late stages.

An analytical quality by design approach towards a simple and novel HPLC-UV method for quantification of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline.

N-acetyl-seryl-aspartyl-lysyl proline (Ac-SDKP) is a tetrapeptide possessing anti-fibrotic, angiogenic, anti-inflammatory, anti-apoptotic, and immunomodulatory properties. Currently, the main method to quantify the peptide is liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA), both of which are labour intensive and require expensive equipment and consumables. Furthermore, these techniques are generally utilised to detect very low or trace concentrations, such as in biological samples. The use of high concentrations of analyte might overload the extraction column or the separation column in LC-MS/MS or the ELISA plates, so the response could be a non-linear relationship at high analyte concentrations. Thus, they are not ideal for formulation development where detection of dose-equivalent concentrations is typically required. Therefore, a cost-effective, simple, and accurate quantification method for the peptide at a higher concentration needs to be developed. In this study, a simple and novel HPLC-UV method is proposed and validated using an Analytical Quality by Design (AQbD) approach. The method is first screened and optimised using chromatographic responses including capacity factor, resolution, tailing factor, and theoretical plate counts, fulfilling the International Council for Harmonisation (ICH) Q2 (R1) guidelines. The resultant optimised chromatography conditions utilised 10 mM phosphate buffer at pH 2.5 and acetonitrile as mobile phases, starting at 3% (v/v) acetonitrile and 97% (v/v) buffer and increasing to 9.7% (v/v) acetonitrile and 90.3% (v/v) buffer over 15 min at a flow rate of 1 mL/min at the column temperature of 25 °C. The injection volume is set at 10 µL and the VWD detector wavelength is 220 nm. The method established is suitable for detecting the peptide at a relatively high concentration, with a quantifiable range from 7.8 µg/mL to 2.0 mg/mL. In addition, the use of a relatively simple HPLC-UV approach could significantly reduce costs and allow easier access to quantify the peptide concentration. A limitation of this method is lower sensitivity compared with using LC-MS/MS and ELISA methods but running costs are lower and the methodology is simpler. The method is capable to quantify the peptide in various tested matrix solutions, with successful quantitation of the peptide in samples obtained from in vitro drug release study in PBS and from a chitosan-TPP nanogels formulation. Therefore, the method developed here offers a complementary approach to the existing quantification methods, quantifying this peptide at increased concentrations in simple to intermediately complex matrix solutions, such as HBSS, DMEM and FluoroBrite cell culture media.

[Regulatory effect of Ac-SDKP on phosphorylated heat shock protein 27/SNAI1 pathway in silicotic rats].

Objective: To study the effect of anti-fibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on phosphorylated heat shock protein 27 (P-HSP27) and zinc finger family transcriptional repressor 1 (SNAI1) expression to explore the anti-silicosis fibrosis effect of Ac-SDKP. Methods: In December 2014, the rat silicosis animal model was prepared by one-time bronchial infusion of silicon dioxide (SiO(2)) dust. 80 SPF healthy adult Wistar rats were selected, and the rats were divided into 8 groups according to the random number table method, 10 in each group. Model control group for 4 weeks (feeding for 4 weeks) , model control group for 8 weeks (feeding for 8 weeks) : bronchial perfusion with normal saline 1.0 ml per animal. Silicosis model group for 4 weeks (feeding for 4 weeks) and silicosis model group for 8 weeks (feeding for 8 weeks) : bronchial perfusion of 50 mg/ml SiO(2) suspension 1.0 ml per animal. Ac-SDKP administration group for 4 weeks (feeding for 4 weeks) , Ac-SDKP administration group for 8 weeks (feeding for 8 weeks) : Ac-SDKP 800 µg·kg(-1)·d(-1) was administered by intraperitoneal pump. Ac-SDKP preventive treatment group: 48 h after Ac-SDKP 800 µg·kg(-1)·d(-1) administration, bronchial perfusion of SiO(2) suspension 1.0 ml per animal, raised for 8 weeks. Ac-SDKP anti-fibrosis treatment group: after bronchial perfusion of 1.0 ml of SiO(2) suspension for 4 weeks, Ac-SDKP 800 µg·kg(-1)·d(-1) was administered for 4 weeks. Western blotting was used to detect the expression of P-HSP27, SNAI1, α-smooth muscle actin (α-SMA) , and collage typeⅠ and Ⅲ in each group. The expression of P-HSP27 and SNAI1 was detected by immunohistochemistry, and the co-localized expression of P-HSP27 and α-SMA was detected by laser confocal microscopy. Results: Compared with the model control group, the expressions of P-HSP27, SNAI1, α-SMA, and collage typeⅠ and Ⅲ in the silicosis fibrosis area of the rats in the silicosis model group were enhanced, and the differences were statistically significant (P<0.05) . After Ac-SDKP intervention, compared with silicosis model group for 8 weeks, the expressions of P-HSP27, SNAI1 α-SMA, and collage typeⅠ and Ⅲ in the Ac-SDKP preventive and anti-fibrosis treatment groups were significantly decreased, and the differences were statistically significant (P<0.05) . However, the expressions of P-HSP27 SNAI1, and collage typeⅠ and Ⅲ between the Ac-SDKP administration group and the model control group did not change significantly, and the differences were not statistically significant (P>0.05) . Laser confocal results showed that the positive cells expressing P-HSP27 and α-SMA in the lung tissue of the silicosis model group were more than those in the model control group. Compared with the silicosis model group, the Ac-SDKP prevention and anti-fibrosis treatment groups expressing the positive cells of P-HSP27 and α-SMA decreased. Compared with the model control group for 8 weeks, there were some double-positive cells expressing P-HSP27 and α-SMA in the nodules of the silicosis model group for 8 weeks. Conclusion: Ac-SDKP may play an anti-silicic fibrosis effect by regulating the P-HSP27/SNAI1 pathway.

Ac-SDKP peptide improves functional recovery following spinal cord injury in a preclinical model.

Damage to the spinal cord triggers a local complex inflammatory reaction that results in irreversible impairments or complete loss of motor function. The evidence suggested that inhibiting the pro-inflammatory macrophage/microglia (M1 subsets) and stimulating the anti-inflammatory macrophage/microglia (M2 subsets) are potential strategies for the treatment of neuroinflammation-related diseases. We evaluated the potentially protective effect of Ac-SDKP as an endogenous tetrapeptide on rat spinal cord injury (SCI). Wistar rats were subjected to a weight-drop contusion model and were treated with Ac-SDKP (0.8 mg/kg) given subcutaneously once a day for 7 days starting at two clinically relevant times, at 2 h or 6 h post-injury. The effect of Ac-SDKP was assessed by motor functional analysis, real-time PCR (CD86 and CD206 mRNA), western blot (caspase-3), ELISA (TNF-a, IL-10), and histological analysis (toluidine blue staining). Ac-SDKP improved locomotor recovery and rescue motor neuron loss after SCI. Moreover, a decreased in TNF-a level as well as caspase 3 protein levels occurred in the lesion epicenter of the spinal cord following treatment. In addition, CD206 mRNA expression level increased significantly in Ac-SDKP treated rats compared with SCI. Together these data suggest that Ac-SDKP might be a novel immunomodulatory drug. It may be beneficial for the treatment of SCI with regards to increasing CD206 gene expression and suppress inflammatory cytokine to improve motor function and reducing histopathological lesion.

Regulatory effect of Ac-SDKP on phosphorylated heat shock protein 27/SNAI1 pathway in silicotic rats / 中华劳动卫生职业病杂志

Objective: To study the effect of anti-fibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on phosphorylated heat shock protein 27 (P-HSP27) and zinc finger family transcriptional repressor 1 (SNAI1) expression to explore the anti-silicosis fibrosis effect of Ac-SDKP. Methods: In December 2014, the rat silicosis animal model was prepared by one-time bronchial infusion of silicon dioxide (SiO(2)) dust. 80 SPF healthy adult Wistar rats were selected, and the rats were divided into 8 groups according to the random number table method, 10 in each group. Model control group for 4 weeks (feeding for 4 weeks) , model control group for 8 weeks (feeding for 8 weeks) : bronchial perfusion with normal saline 1.0 ml per animal. Silicosis model group for 4 weeks (feeding for 4 weeks) and silicosis model group for 8 weeks (feeding for 8 weeks) : bronchial perfusion of 50 mg/ml SiO(2) suspension 1.0 ml per animal. Ac-SDKP administration group for 4 weeks (feeding for 4 weeks) , Ac-SDKP administration group for 8 weeks (feeding for 8 weeks) : Ac-SDKP 800 μg·kg(-1)·d(-1) was administered by intraperitoneal pump. Ac-SDKP preventive treatment group: 48 h after Ac-SDKP 800 μg·kg(-1)·d(-1) administration, bronchial perfusion of SiO(2) suspension 1.0 ml per animal, raised for 8 weeks. Ac-SDKP anti-fibrosis treatment group: after bronchial perfusion of 1.0 ml of SiO(2) suspension for 4 weeks, Ac-SDKP 800 μg·kg(-1)·d(-1) was administered for 4 weeks. Western blotting was used to detect the expression of P-HSP27, SNAI1, α-smooth muscle actin (α-SMA) , and collage typeⅠ and Ⅲ in each group. The expression of P-HSP27 and SNAI1 was detected by immunohistochemistry, and the co-localized expression of P-HSP27 and α-SMA was detected by laser confocal microscopy. Results: Compared with the model control group, the expressions of P-HSP27, SNAI1, α-SMA, and collage typeⅠ and Ⅲ in the silicosis fibrosis area of the rats in the silicosis model group were enhanced, and the differences were statistically significant (P<0.05) . After Ac-SDKP intervention, compared with silicosis model group for 8 weeks, the expressions of P-HSP27, SNAI1 α-SMA, and collage typeⅠ and Ⅲ in the Ac-SDKP preventive and anti-fibrosis treatment groups were significantly decreased, and the differences were statistically significant (P<0.05) . However, the expressions of P-HSP27 SNAI1, and collage typeⅠ and Ⅲ between the Ac-SDKP administration group and the model control group did not change significantly, and the differences were not statistically significant (P>0.05) . Laser confocal results showed that the positive cells expressing P-HSP27 and α-SMA in the lung tissue of the silicosis model group were more than those in the model control group. Compared with the silicosis model group, the Ac-SDKP prevention and anti-fibrosis treatment groups expressing the positive cells of P-HSP27 and α-SMA decreased. Compared with the model control group for 8 weeks, there were some double-positive cells expressing P-HSP27 and α-SMA in the nodules of the silicosis model group for 8 weeks. Conclusion: Ac-SDKP may play an anti-silicic fibrosis effect by regulating the P-HSP27/SNAI1 pathway.

Investigating the antifibrotic potential of N-acetyl seryl-aspartyl-lysyl-proline sequence peptides.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a physiological antifibrotic peptide that is hydrolysed by angiotensin I-converting enzyme (ACE). The beneficial antifibrotic effects of ACE inhibitors have been attributed, in part, to its inhibition of Ac-SDKP cleavage. There is indirect evidence that the SDK fragment of Ac-SDKP is the main component required for its antiproliferative action. However, the exact component of the physiological peptide that is responsible for its antifibrotic effect has yet to be determined. Ac-SDKP-derived analogues that are resistant to ACE degradation may provide a new avenue for fibrosis therapy. We tested the antifibrotic potential of various Ac-SDKP peptide sequences and an analogue resistant to ACE degradation in lung fibroblasts. We investigated the contribution and molecular mechanism of action of the amino acid residues in the Ac-SDKP sequence to its antifibrotic effects, and the effects of Ac-SDKP peptides in the prevention of collagen deposition in cells. The Ac-DKP fragment moderately inhibited endothelin-1 (ET-1) mediated transforming growth factor-ß (TGF- ß) expression, and could be slowly cleaved by ACE, revealing a different sequence requirement for the antifibrotic action of Ac-SDKP. The Ac-SDψKP analogue (where the peptide bond between the aspartate and lysine is reduced) inhibited TGF-ß/small mother against decapentaplegic (Smad)-3 signalling and collagen deposition. The Ac-SDKP peptide, in combination with ACEi, demonstrated a greater inhibition of hydroxyproline as compared to Ac-SDKP alone.

Renin-angiotensin system and inflammation update.

The most classical view of the renin-angiotensin system (RAS) emphasizes its role as an endocrine regulator of sodium balance and blood pressure. However, it has long become clear that the RAS has pleiotropic actions that contribute to organ damage, including modulation of inflammation. Angiotensin II (Ang II) activates angiotensin type 1 receptors (AT1R) to promote an inflammatory response and organ damage. This represents the pathophysiological basis for the successful use of RAS blockers to prevent and treat kidney and heart disease. However, other RAS components could have a built-in capacity to brake proinflammatory responses. Angiotensin type 2 receptor (AT2R) activation can oppose AT1R actions, such as vasodilatation, but its involvement in modulation of inflammation has not been conclusively proven. Angiotensin-converting enzyme 2 (ACE2) can process Ang II to generate angiotensin-(1-7) (Ang-(1-7)), that activates the Mas receptor to exert predominantly anti-inflammatory responses depending on the context. We now review recent advances in the understanding of the interaction of the RAS with inflammation. Specific topics in which novel information became available recently include intracellular angiotensin receptors; AT1R posttranslational modifications by tissue transglutaminase (TG2) and anti-AT1R autoimmunity; RAS modulation of lymphoid vessels and T lymphocyte responses, especially of Th17 and Treg responses; interactions with toll-like receptors (TLRs), programmed necrosis, and regulation of epigenetic modulators (e.g. microRNAs and bromodomain and extraterminal domain (BET) proteins). We additionally discuss an often overlooked effect of the RAS on inflammation which is the downregulation of anti-inflammatory factors such as klotho, peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), transient receptor potential ankyrin 1 (TRPA1), SNF-related serine/threonine-protein kinase (SNRK), serine/threonine-protein phosphatase 6 catalytic subunit (Ppp6C) and n-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Both transcription factors, such as nuclear factor κB (NF-κB), and epigenetic regulators, such as miRNAs are involved in downmodulation of anti-inflammatory responses. A detailed analysis of pathways and targets for downmodulation of anti-inflammatory responses constitutes a novel frontier in RAS research.

Angiotensin-converting enzyme inhibitors increase anti-fibrotic biomarkers in African Americans with left ventricular hypertrophy.

Angiotensin-converting enzyme inhibitors (ACEi) are part of the indicated treatment in hypertensive African Americans. ACEi have blood pressure-independent effects that may make them preferred for certain patients. We aimed to evaluate the impact of ACEi on anti-fibrotic biomarkers in African American hypertensive patients with left ventricular hypertrophy (LVH). We conducted a post hoc analysis of a randomized controlled trial in which hypertensive African American patients with LVH and vitamin D deficiency were randomized to receive intensive antihypertensive therapy plus vitamin D supplementation or placebo. We selected patients who had detectable lisinopril (lisinopril group) in plasma using liquid-chromatography/mass spectrometry analysis and compared them to subjects who did not (comparison group) at the one-year follow-up. The pro-fibrotic marker type 1 procollagen C-terminal propeptide (PICP) and the anti-fibrotic markers matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinases 1 (TIMP-1), telopeptide of collagen type I (CITP), and N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) peptide were measured. Sixty-six patients were included, and the mean age was 46.2 ± 8 years. No difference was observed in the number and intensity of antihypertensive medications prescribed in each group. Patients with detectable lisinopril had lower blood pressure than those in the comparison group. The anti-fibrotic markers Ac-SDKP, MMP-1, and MMP-1/TIMP-1 ratio were higher in patients with detectable ACEi (all p < .05). In a model adjusted for systolic blood pressure, MMP-1/TIMP-1 (p = .02) and Ac-SDKP (p < .001) levels were associated with lisinopril. We conclude that ACEi increase anti-fibrotic biomarkers in hypertensive African Americans with LVH, suggesting that they may offer added benefit over other agents in such patients.

A long-acting isomer of Ac-SDKP attenuates pulmonary fibrosis through SRPK1-mediated PI3K/AKT and Smad2 pathway inhibition.

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening lung disease with a poor prognosis. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a critical negative regulator of fibrosis development. However, it's extremely short half-life greatly limits its applications. Previously, we reported an Ac-SDKP analog peptide in which Asp and Lys residues were replaced with D-amino acids (Ac-SDD KD P). Ac-SDD KD P exhibits better resistance to angiotensin-1-converting enzyme (ACE)-mediated degradation and a longer half-life than Ac-SDKP in rat and human sera. The objective of this study was to explore the potential application of Ac-SDD KD P for the treatment of IPF and to clarify the underlying mechanisms. We found that Ac-SDD KD P exerted similar antifibrotic effects as Ac-SDKP on human fetal lung fibroblast-1 (HFL-1) proliferation, α-smooth muscle actin (α-SMA), collagen I and collagen III expression, and Smad-2 phosphorylation in vitro. In vivo, Ac-SDD KD P exhibited significantly greater protective effects against bleomycin-induced pulmonary fibrosis than Ac-SDKP in mice. α-SMA, CD45, collagen I and collagen III expression, and Smad-2 phosphorylation were significantly decreased in the lungs of Ac-SDD KD P-treated but not Ac-SDKP-treated mice. Furthermore, a pull-down experiment was used to screen for molecules that interact with Ac-SDKP. Co-immunoprecipitation (Co-IP) and computer-based molecular docking experiments demonstrated an interaction between Ac-SDKP or Ac-SDD KD P (Ac-SDKP/Ac-SDD KD P) and serine/arginine-rich protein-specific kinase 1 (SRPK1) that caused inhibition SRPK1-mediated phosphatidylinositol-3 kinase/ serine/threonine kinase (PIK3/AKT) signaling pathway activation and Smad2 phosphorylation and thereby attenuated lung fibrosis. Our data suggest that long-acting Ac-SDD KD P may potentially be an effective drug for the treatment of pulmonary fibrosis. The interacting molecule and antifibrotic mechanism of Ac-SDKP/Ac-SDD KD P were also identified, providing an experimental and theoretical foundation for the clinical application of the drug.

Synthesis and Identification of a Novel Peptide, Ac-SDK (Biotin) Proline, That Can Elicit Anti-Fibrosis Effects in Rats Suffering from Silicosis.

BACKGROUND: N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a short peptide with an anti-silicosis effect. However, the short biological half-life and low plasma concentration of Ac-SDKP hamper discovery of specific targets in organisms and reduce the anti-silicosis effect. A novel peptide, Ac-SDK (biotin) proline, termed "Ac-B", with anti-fibrotic properties was synthesized. METHODS: Ac-B was detected quantitatively by high-performance liquid chromatography. Phagocytosis of Ac-B by the alveolar epithelial cell line A549 was investigated by confocal laser scanning microscopy and flow cytometry. To further elucidate the cellular-uptake mechanism of Ac-B, chemical inhibitors of specific uptake pathways were used. After stimulation with transforming growth factor-ß1, the effects of Ac-B on expression of the myofibroblast marker vimentin and accumulation of collagen type I in A549 cells were analyzed by Western blotting. Sirius Red staining and immunohistochemical analyses of the effect of Ac-B on expression of α-smooth muscle actin (SMA) in a rat model of silicosis were undertaken. RESULTS: Ac-B had good traceability during the uptake, entry, and distribution in cells. Ac-B treatment prevented an increase in α-SMA expression in vivo and in vitro and was superior to that of Ac-SDKP. Caveolae-mediated uptake of Ac-B by A549 cells led to achieving anti-epithelial-mesenchymal transformation (EMT) effects. CONCLUSION: Ac-B had an anti-fibrotic effect and could be a promising agent for the fibrosis observed in silicosis in the future.

N-Acetyl-Seryl-Asparyl-Lysyl-Proline regulates lung renin angiotensin system to inhibit epithelial-mesenchymal transition in silicotic mice.

Silicosis is a major public health concern with various contributing factors. The renin-angiotensin system (RAS)is a critical regulator in the pathogenesis of this disease. We focused on two key RAS enzymes, angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2), to elucidate the activation of the ACE-angiotensin II (Ang II)-angiotensin II receptor 1 (AT1) axis and the inhibition of the ACE2-angiotensin-(1-7) [Ang-(1-7)]-Mas receptor axis in C57BL/6mice following SiO2 treatment. Silica exposure caused nodule formation, pulmonary interstitial fibrosis, epithelial-mesenchymal transition (EMT), abnormal deposition of extracellular matrix, and impaired lung function in mice. These effects were attenuated by the inhibition of ACE (captopril), blockade of the AT1(losartan), or systemic knockdown of the Ace gene. These effects were exacerbated by the inhibition of ACE2 (MLN-4760), blockade of the Mas (A779), or knockdown of the Ace2 gene. N-Acetyl-Seryl-Asparyl-Lysyl-Proline (Ac-SDKP), an anti-fibrotic peptide, ameliorated the silica-exposure-induced pathological changes by targeting the RAS system by activating the protective ACE2-Ang-(1-7)-Mas axis and inhibiting the deleterious ACE-Ang II-AT1 axis, thereby exerting a protective effect. This was confirmed in mouse lung type II epithelial cells (MLE-12) pretreated with Ang II and/or gene silencing separately targeting Ace and Ace2.The effects of Ac-SDKP were similar to those produced by Ace gene silencing and were partly attenuated by Ace2 deficiency. These findings suggested that RAS plays critical roles in the pathomechanism of silicosis fibrosis and that Ac-SDKP regulates lung RAS to inhibit EMT in silicotic mice and MLE-12 cells.

Engineered M13 Peptide Carrier Promotes Angiogenic Potential of Patient-Derived Human Cardiac Progenitor Cells and In Vivo Engraftment.

BACKGROUND: Despite promising advances in stem cell-based therapy, the treatment of ischemic cardiovascular diseases remains a big challenge due to both the insufficient in vivo viability of transplanted cells and poor angiogenic potential of stem cells. The goal of this study was to develop therapeutic human cardiac progenitor cells (hCPCs) for ischemic cardiovascular diseases with a novel M13 peptide carrier. METHOD: In this study, an engineered M13 peptide carrier was successfully generated using a QuikChange Kit. The cellular function of M13 peptide carrier-treated hCPCs was assessed using a tube formation assay and scratch wound healing assay. The in vivo engraftment and cell survival bioactivities of transplanted cells were demonstrated by immunohistochemistry after hCPC transplantation into a myocardial infarction animal model. RESULTS: The engineered M13RGD+SDKP peptide carrier, which expressed RGD peptide on PIII site and SDKP peptide on PVIII site, did not affect morphologic change and proliferation ability in hCPCs. In contrast, hCPCs treated with M13RGD+SDKP showed enhanced angiogenic capacity, including tube formation and migration capacity. Moreover, transplanted hCPCs with M13RGD+SDKP were engrafted into the ischemic region and promoted in vivo cell survival. CONCLUSION: Our present data provides a promising protocol for CPC-based cell therapy via short-term cell priming of hCPCs with engineered M13RGD+SDKP before cell transplantation for treatment of cardiovascular disease.