KPV Research & Studies

Cardiovascular disease (CVD) is a leading cause of death globally, and vascular calcification (VC) is an important independent risk factor for predicting CVD. Currently, there are no established therapeutic strategies for the treatment of VC. Although recognized combination therapies of nanomedicines can provide effective strategies for disease treatment, the clinical application of nanomedicines is limited because of their complex preparation processes, low drug loading rates, and unpredictable safety risks. Thus, developing a simple, efficient, and safe nanodrug to simultaneously regulate inflammation and autophagy may be a promising strategy for treating VC. Herein, an anti-inflammatory peptide (lysine-proline-valine peptides, KPV) and the autophagy activator rapamycin (RAPA) are self-assembled to form new carrier-free spherical nanoparticles (NPs), which shows good stability and biosafety. In vivo and in vitro, KPV-RAPA NPs significantly inhibit VC in mice compared to the other treatment groups. Mechanistically, KPV-RAPA NPs inhibit inflammatory responses and activated autophagy. Therefore, this study indicates that the new carrier-free KPV-RAPA NPs have great potential as therapeutic agents for VC combination therapy, which can promote the development of nanodrugs for VC.

Inflammatory bowel disease (IBD), as a chronic and recurrent inflammatory bowel diseases with limited therapeutic outcomes, is characterized by immune disorders and intestinal barrier dysfunction. Currently, the most medications used to cure IBD in clinic just temporarily induce and maintain remission with poor response rates and limited outcomes. Therefore, it is urgently necessary to develop an appropriate therapeutic candidate with preferable efficacy and less adverse reaction for curing IBD.

Ulcerative colitis (UC) usually occurs in the superficial mucosa of the colorectum. Here, a double-network hydrogel (PMSP) was constructed from maleimided γ-polyglutamic acid and thiolated γ-polyglutamic acid through crosslinking of thiol-maleimide and self-oxidized thiols. PMSP with a negative charge specifically adhered to the inflamed mucosa with positively charged proteins rather than to the healthy mucosa. PMSP exhibited good mechanical strength with storage modulus (G') of 17.6 Pa and a linear viscoelastic region (LVR) of 107.2% strain. Moreover, PMSP showed a stronger bio-adhesive force toward the inflamed tissue-mimicking substrate than toward its healthy counterpart. In vivo imaging confirmed that PMSP specifically adhered to the inflamed colonic mucosa of rats with TNBS-induced UC. KPV (Lys-Pro-Val) as a model drug was easily captured by PMSP through electrostatic interactions, thus retaining its bioactivity for a longer time under high temperature conditions. Moreover, the alleviating effect of KPV on rats with TNBS-induced colitis was significantly improved by PMSP after intracolonic administration. The epithelial barrier of the colon also effectively recovered following PMSP-KPV treatment. PMSP-KPV also modulated the gut flora, markedly augmenting the abundance of beneficial microorganisms in gut homeostasis. The mechanism by which PMSP-KPV induces a therapeutic effect may be associated with the inhibition of oxidative stress. Conclusively, the PMSP hydrogel seems to be a promising rectal delivery system for the therapy of UC. STATEMENT OF SIGNIFICANCE: Ulcerative colitis (UC) is a chronic and relapsing disease of the gastrointestinal tract. A key therapeutic approach to treat UC is to repair the mucosal barriers. Here, a double-network hydrogel (PMSP) was constructed from maleimided and thiolated γ-polyglutamic acid through crosslinking of thiol-maleimide and self-oxidized thiols. The negatively charged PMSP specifically adhered to the inflamed colon rather than its healthy counterpart and was retained for a longer time. KPV as a model drug was easily captured by PMSP, which provided better stability to KPV when exposed to high temperature of 50 °C. The epithelial mucosal barrier of the colon was effectively recovered by the rectal administration of PMSP-KPV to rats with TNBS-induced UC. Moreover, PMSP-KPV modulated the gut flora of colitic rats, markedly augmenting the abundance of beneficial microorganisms. Conclusively, PMSP seems to be a promising rectal delivery system for UC therapy.

The general treatment of diabetic wound was use of wound dressings to absorb excess exudate. However, traditional wound dressings neither mimic the skin-like properties nor easily be withdrawn from the wound. Herein, the skin-adaptive three-layered films (AGB) dressing has been designed by alternatively depositing phenylboronic acid-grafted γ-PGA (PBA-PGA) and polyvinyl alcohol (PVA). The thickness of AGB film was only 479 μm and its flexibility was obviously strengthen by the boronic ester cross-linking. Besides, the dry AGB film was conveniently adhered to the fresh wound, where its adhesive force reached to 1267 ± 330 mN. Moreover, the adhered AGB film was easily peeled without any second damage after hydration. An anti-inflammatory tripeptide (KPV) and epidermal growth factor (EGF) as biologic factors were respectively encapsulated in the bottom layer and the middle-top two layers of AGB film. KPV was firstly released within 3 day and EGF was subsequently released in a glucose-responsive manner. AGB film containing KPV and EGF (K-E-AGB) could significantly improve the repair rate of full-thickness skin wound on diabetic mice. The mechanism of wound healing was associated with inflammatory inhibition, angiogenesis and collagen deposition. Collectively, skin-adaptive film may be a promising dressing as delivery of biologic factors for the chronic wound.

To effectively alleviate acute severe ulcerative colitis (ASUC), we developed a colon-specific delivery system-PLGA-KPV/MMT/CS multifunctional medicinal nanoparticles loaded with cyclosporine A (CyA). The lysine-proline-valine (KPV) tripeptide, which possesses anti-inflammatory properties and high affinity to peptide transporter 1 (PepT1), can target therapy-related cells (colonic epithelial cells and macrophages) via overexpression of PepT1. Montmorillonite (MMT)/chitosan (CS) coating can reduce CyA leakage in the upper gastrointestinal tract (GIT) and enhance nanoparticle adhesion to the inflamed colon. The bio-distribution demonstrated that nanoparticles can specifically accumulate in the inflamed tissues and can be retained for up to 36 h. After being treated with the CyA-PLGA-KPV/MMT/CS nanoparticles (PKMCN), the mice with DSS-induced ulcerative colitis exhibited significant improvements in body weight, colon length, and disease activity index. Moreover, biochemistry and immunohistochemical analysis showed that the PKMCN treatment group performed as well as the healthy group. Intriguingly, PKMCN without CyA also presented marked therapeutic effects. Our results suggested that PKMCN could be a promising drug delivery system for ASUC therapy by targeting inflamed cells, prolonging curative time, and mitigating colitis.

Overcoming adverse effects and selectively delivering drug to target cells are two major challenges in the treatment of ulcerative colitis (UC). Lysine-proline-valine (KPV), a naturally occurring tripeptide, has been shown to attenuate the inflammatory responses of colonic cells. Here, we loaded KPV into hyaluronic acid (HA)-functionalized polymeric nanoparticles (NPs). The resultant HA-KPV-NPs had a desirable particle size (∼272.3 nm) and a slightly negative zeta potential (∼-5.3 mV). These NPs successfully mediated the targeted delivery of KPV to key UC therapy-related cells (colonic epithelial cells and macrophages). In addition, these KPV-loaded NPs appear to be nontoxic and biocompatible with intestinal cells. Intriguingly, we found that HA-KPV-NPs exert combined effects against UC by both accelerating mucosal healing and alleviating inflammation. Oral administration of HA-KPV-NPs encapsulated in a hydrogel (chitosan/alginate) exhibited a much stronger capacity to prevent mucosa damage and downregulate TNF-α, thus they showed a much better therapeutic efficacy against UC in a mouse model, compared with a KPV-NP/hydrogel system. These results collectively demonstrate that our HA-KPV-NP/hydrogel system has the capacity to release HA-KPV-NPs in the colonic lumen and that these NPs subsequently penetrate into colitis tissues and enable KPV to be internalized into target cells, thereby alleviating UC.

The inflammatory activity of ulcerative colitis plays an important role in the medical treatment. However, accurate and real-time monitoring of the colitis activity with noninvasive bioimaging method is still challenging, especially in distinguishing between chronic and acute colitis. As a good receptor, the oligopeptide transporter (PepT1) is overexpressed in the colonic epithelial cells of chronic ulcerative colitis, which can deliver tripeptide KPV (Lys-Pro-Val, the C-terminal sequence of α-MSH) into cytosol in the intestine. Herein, we report a PepT1 peptide receptor-targeted fluorescent probe, dicyanomethylene-4H-pyran (DCM)-KPV, with the strategy of conjugating the KPV into the DCM chromophore. The diagnostic fluorescent probe bestows a specific receptor-targeted interaction with PepT1 through the KPV moiety, possessing several beneficial characteristics, such as efficient long emission, low photobleaching, negligible cytotoxicity, and high cytocompatibility in living cells. We build the overexpressed PepT1 on the cytomembrane of ulcerative colitis model Caco-2 cell as the efficient receptor to accumulate the targeted tripeptide KPV in the cytoplasm and nucleus. With the co-localization of DCM-KPV and the DNA-specific fluorophore, DAPI, the specifically long emission from chromophore DCM and efficient receptor-targeted peptide KPV, the fluorescent probe of DCM-KPV makes a breakthrough to the direct noninvasive observation of the accumulation in colon inflammation regions via intestinal mucosa, even successfully distinguishing the chronic, acute ulcerative colitis and normal groups. Compared with the traditional unenhanced magnetic resonance imaging and hematoxylin and eosin (H&E) staining, we make full use of exploiting the specific target-receptor interaction between the tripeptide unit, KPV, and the oligopeptide transporter, PepT1, for sensing selectivity. The desirable diagnostic ability of DCM-KPV can guarantee the real-time tracking and visualization of the role of intracellular KPV on ulcerative colitis, which provides an alternative to replace the time-consuming and tissue sampling-invasive H&E staining diagnosis.

The crude polysaccharide (KPV-0) isolated from Korean persimmon vinegar was fractionated using gel filtration chromatography to enhance the immunostimulatory activity and to identify the structural features of active fraction. Among three fractions, KPV-I obtained in a void volume, demonstrated the potent production of macrophage-stimulating mediators, including tumor necrosis factor-α, interleukin (IL)-6, IL-12, and nitric oxide. KPV-I showed a combined single peak with high molecular weight of 55,000Da by high performance size exclusion chromatography. Component sugar analysis revealed that KPV-I contained mainly of arabinose, mannose, galactose, rhamnose and galacturonic acid. Single radial gel diffusion assay using β-glucosyl Yariv reagent showed that KPV-I contained arabinogalactan protein with 13.7%. Methylation analysis indicated that KPV-I contained 21 kinds of neutral glycosidic linkages, which seemed to be composed three kinds of polysaccharide; that is a rhamnogalacturonan-I (65-70%) derived from persimmon as a raw material, a mannan (20-25%) derived from fermentation-associated microorganisms, and a linear glucans (less than 10%). In conclusion, polysaccharide isolated from persimmon vinegar could augment the macrophage stimulation, and a large amounts of RG-I polysaccharide derived from persimmon is likely a crucial role in expression of the activity in persimmon vinegar.

A simple, sensitive and stability-indicating high-performance liquid chromatographic (HPLC) assay method was developed and validated for a bioactive peptide, lysine-proline-valine (KPV) in aqueous solutions and skin homogenates. Chromatographic separation was achieved on a reversed phase Phenomenex C18 column (4.6 × 250 mm, packed with 5 µm silica particles) with a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). The proposed HPLC method was validated with respect to accuracy, precision, linearity, repeatability, limit of detection (LOD) and limit of quantitation (LOQ). The calibration curve was linear with a correlation coefficient (r) of 0.9999. Relative standard deviation values of accuracy and precision experiments were <2. The LOD and LOQ of KPV were 0.01 and 0.25 µg/mL, respectively. Under stress conditions (acid, alkali and hydrogen peroxide) KPV yielded lys-pro-diketopiperazine as major degradation product, which was identified by flow injection MS analysis. The developed HPLC method was found to be efficient in separating the active peptide from its degradation products generated under various stress conditions. Also, the validated method was able to separate KPV from other peaks arising from endogenous components of the skin homogenate.

Chemokine signaling from airway epithelium regulates macrophage recruitment to the lung in inflammatory diseases such as asthma. This study investigates the mechanism by which the α-melanocyte stimulating hormone-derived tripeptide, KPV, and the agonist of the dominant melanocortin receptor in airway epithelium (MC3R), γ-melanocyte stimulating hormone (γ-MSH), suppress inflammation in immortalised human bronchial airway epithelium.

One of the challenges to treating inflammatory bowel disease (IBD) is to target the site of inflammation. We engineered nanoparticles (NPs) to deliver an anti-inflammatory tripeptide Lys-Pro-Val (KPV) to the colon and assessed its therapeutic efficacy in a mouse model of colitis.

During the last two decades a significant number of investigations has established the fact that α-Melanocyte-stimulating hormone (α-MSH) is a potent anti-inflammatory mediator. The anti-inflammatory effects of α-MSH can be elicited via melanocortin receptors (MC-Rs) broadly expressed in a number of tissues ranging from the central nervous system to cells of the immune system and on resident somatic cells of peripheral tissues. α-MSH affects various pathways regulating inflammatory responses such as NF-κB activation, expression of adhesion molecules, inflammatory cytokines, chemokine receptors, T-cell proliferation and activity and inflammatory cell migration. In vivo α-MSH has been shown to be anti-inflammatory as well in animal models of fever, irritant and allergic contact dermatitis, cutaneous vasculitis, fibrosis, in ocular, gastrointestinal, brain and allergic airway inflammation and arthritis. A broad range of effects of α-MSH exerted beyond the field of inflammation, its pigmentory capacity being only the most visible aspect, has been one of the major impediments limiting the use of α-MSH in human inflammatory disorders. Interestingly KPV, C-terminal tripeptide of α-MSH, which lacks the entire sequence motif required for binding to any of the known MC-Rs, retains almost all of the anti-inflammatory capacity of the full hormone, but in its activities display a lack of any pigmentory action. While the exact signaling mechanism utilized by KPV and related peptides currently is unknown it has been demonstrated already that significant similarities between anti-inflammatory signaling of α-MSH and those short peptides exist. These α-MSH related tripeptides thus may be useful alternatives for anti-inflammatory peptide therapy. KdPT, a derivative of KPV corresponding to IL-1β(193-195), currently is emerging as another tripeptide with potent anti-inflammatory effects. A more limited spectrum of biologic activities, potentially advantageous physicochemical, pharmacokinetic and pharmacodynamic properties as well as the expectation of low costs for pharmaceutical production make these agents interesting candidates for the treatment of immune-mediated inflammatory skin and bowel diseases, allergic asthma and arthritis.

Alpha-MSH is a tridecapeptide derived from proopiomelanocortin. Many studies over the last few years have provided evidence that alpha-MSH has potent protective and antiinflammatory effects. These effects can be elicited via centrally expressed melanocortin receptors that orchestrate descending neurogenic antiinflammatory pathways. alpha-MSH can also exert antiinflammatory and protective effects on cells of the immune system and on peripheral nonimmune cell types expressing melanocortin receptors. At the molecular level, alpha-MSH affects various pathways implicated in regulation of inflammation and protection, i.e., nuclear factor-kappaB activation, expression of adhesion molecules and chemokine receptors, production of proinflammatory cytokines and mediators, IL-10 synthesis, T cell proliferation and activity, inflammatory cell migration, expression of antioxidative enzymes, and apoptosis. The antiinflammatory effects of alpha-MSH have been validated in animal models of experimentally induced fever; irritant and allergic contact dermatitis, vasculitis, and fibrosis; ocular, gastrointestinal, brain, and allergic airway inflammation; and arthritis, but also in models of organ injury. One obstacle limiting the use of alpha-MSH in inflammatory disorders is its pigmentary effect. Due to its preserved antiinflammatory effect but lack of pigmentary action, the C-terminal tripeptide of alpha-MSH, KPV, has been delineated as an alternative for antiinflammatory therapy. KdPT, a derivative of KPV corresponding to amino acids 193-195 of IL-1beta, is also emerging as a tripeptide with antiinflammatory effects. The physiochemical properties and expected low costs of production render both agents suitable for the future treatment of immune-mediated inflammatory skin and bowel disease, fibrosis, allergic and inflammatory lung disease, ocular inflammation, and arthritis.

alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.

In this study, we analyzed the anti-inflammatory effects of alpha-melanocyte stimulating hormone (MSH)11-13 (KPV) in comparison with other MSH peptides in a model of crystal-induced peritonitis. Systemic treatment of mice with KPV, alpha-MSH, the core melanocortin peptide His-Phe-Arg-Trp, and the melanocontin receptor 3/4 agonist Ac-Nle4-c[Asp5,d-Phe7,Lys10]NH2 ACTH4-10 (MTII) but not the selective MC1-R agonist H-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (MS05) resulted in a significant reduction in accumulation of polymorphonuclear leukocyte in the peritoneal cavity. The antimigratory effect of KPV was not blocked by the MC3/4-R antagonist Ac-Nle4-c[Asp5,d-2Nal7,Lys10]NH2 ACTH4-10 (SHU9119). In vitro, macrophage activation, determined as release of KC and interleukin (IL)-1beta was inhibited by alpha-MSH and MTII but not by KPV. Furthermore, macrophage activation by MTII led to an increase in cAMP accumulation, which was attenuated by SHU9119, whereas KPV failed to increase cAMP. The anti-inflammatory properties of KPV were also evident in IL-1beta-induced peritonitis inflammation and in mice with a nonfunctional MC1-R (recessive yellow e/e mice). In conclusion, these data highlight that the C-terminal MSH peptide KPV exhibits an anti-inflammatory effect that is clearly different from that of the core MSH peptides. KPV is unlikely to mediate its effects through melanocortin receptors but is more likely to act through inhibition of IL-1beta functions.

There is a substantial body of evidence that the tridecapeptide alpha-melanocyte-stimulating hormone (alpha-MSH) functions as a mediator of immunity and inflammation. The immunomodulating capacity of alpha-MSH is primarily because of its effects on melanocortin receptor (MC-1R)-expressing monocytes, macrophages, and dendritic cells (DCs). alpha-MSH down-regulates the production of proinflammatory and immunomodulating cytokines (IL-1, IL-6, TNF-alpha, IL-2, IFN-gamma, IL-4, IL-13) as well as the expression of costimulatory molecules (CD86, CD40, ICAM-1) on antigen-presenting DCs. In contrast, the production of the cytokine synthesis inhibitor IL-10 is up-regulated by alpha-MSH. At the molecular level, these effects of alpha-MSH are mediated via the inhibition of the activation of transcription factors such as NFkappaB. Not only alpha-MSH but also its C-terminal tripeptide (alpha-MSH 11-13, KPV) was able to bind to MC-1R and to modulate the function of APCs. In vivo, using a mouse model of contact hypersensitivity (CHS) systemic and topical application of alpha-MSH or KPV inhibited the sensitization and the elicitation phase of CHS and was able to induce hapten-specific tolerance. To investigate the underlying mechanisms of tolerance induction, we have performed in vivo transfer experiments. Treatment of naive mice with bone marrow-derived immature haptenized and alpha-MSH-pulsed DCs resulted in a significant inhibition of CHS. Furthermore, tolerance induction was found to be mediated by the generation of CTLA4(+) and IL-10-producing T lymphocytes. The potent capacity of alpha-MSH to modulate the function of antigen-presenting cells (APCs) has been further supported in another experimental approach. In vitro, by activating APCs, alpha-MSH has been shown to modulate IgE production by IL-4 and anti-CD40 stimulated B lymphocytes. Moreover, in a murine model of allergic airway inflammation, systemic treatment with alpha-MSH resulted in a significant reduction of allergen-specific IgE production, eosinophil influx, and IL-4 production. These effects were mediated via IL-10 production, because IL-10 knockout mice were resistant to alpha-MSH treatment. Therefore, therapeutic application of alpha-MSH or related peptides (KPVs) as well as alpha-MSH/KPV-pulsed DCs may be a useful approach for the treatment of inflammatory, autoimmune, and allergic diseases in the future.

Alpha-melanocyte-stimulating hormone (alpha-MSH), a neuroimmunomodulatory peptide of ancient origin, is known to be involved in the control of host responses. In inflammatory cells, in the periphery and within the central nervous system, alpha-MSH modulates the production and action of proinflammatory cytokines. This broad influence occurs via endogenous alpha-MSH (melanocortin) receptors. The key to this anti-inflammatory influence is inhibition of NF-kappa B. Indeed alpha-MSH inhibits activation of this nuclear factor through preservation of I kappa B alpha, which binds to NF-kappa B and prevents its migration to the nucleus. Cells transfected with alpha-MSH plasmid vector are resistant to challenge with bacterial lipopolysaccharide. The peptide also act on central melanocortin receptors to modulate inflammation in the periphery. In brief, alpha-MSH and certain of its fragments such as alpha-MSH [11-13] KPV modulate inflammation via three general actions: direct actions on peripheral host cells; actions on inflammatory cells within the brain to modulate local reactions; and descending neural anti-inflammatory pathways that control inflammation in peripheral tissues.

The presence of the ancient peptide alpha-melanocyte-stimulating hormone (alpha-MSH) in barrier organs such as gut and skin suggests that this potent anti-inflammatory molecule may be a component of the innate host defense. In tests of antimicrobial activities, alpha-MSH and its fragment KPV showed inhibitory influences against the gram-positive bacterium Staphylococcus aureus and the yeast Candida albicans. Anti-tumor necrosis factor and antimicrobial effects of alpha-MSH suggest that the peptide might likewise reduce replication of human immunodeficiency virus (HIV). Treatment with alpha-MSH reduced HIV replication in chronically and acutely infected human monocytes. At the molecular level, alpha-MSH inhibited activation of the transcription factor NF-kappa B known to enhance HIV expression. alpha-MSH that combines antipyretic, anti-inflammatory, and antimicrobial effects could be useful in the treatment of disorders in which infection and inflammation coexist.