A comparison of the wound healing dynamics in an acute and newly developed chronic wound model using Neutral endopeptidase, an inhibitor of Substance P.

Boodhoo, Kiara (2020-03)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Chronic wounds affect millions of people with diabetes world-wide. Various diabetic wound healing animal models exist, none of which currently replicate chronic wounds in diabetic patients. Animal models that do not closely resemble human conditions delay application of promising therapeutic strategies in clinical settings. Substance P is a neuropeptide that promotes wound healing by binding to neurokinin-1 receptor initiating the inflammatory process. Neutral endopeptidase (NEP) is a zinc metalloprotease that degrades substance P, thereby hindering the healing process. Increased NEP enzymatic activity is evident in chronic wounds of diabetic patients unlike in current animal models. This study aimed to determine the optimum dose of NEP for injection into wound edges in obese pre-diabetic mice (B6.Cg- Lepob/J, ob/ob) in order to create a chronic non-healing wound. Thereafter, the study investigated wound healing dynamics of this chronic non-healing wound compared to an acute wound treated with saline. In addition to a saline control, three concentrations of NEP [low NEP (0.33 mg/μL), medium NEP (0.68 mg/μL) or high NEP (1.02 mg/μL)] were used to obtain a dose-response curve. NEP activities did not differ between treatment groups however, substance P was lower in the wound areas for high NEP compared to low NEP groups. Therefore, less substance P was present to initiate wound healing with high NEP. MMP-9 was higher with high NEP treatment on day 2 compared to saline, low and medium NEP groups. Similarly, the cytokine profile within pooled samples of day 2 wound fluid was lowest with high NEP treatment. Therefore, high NEP application did mimic some characteristics of a chronic wound during the early stages post wounding, including delayed onset of inflammation. When comparing the healing dynamics of this chronic non-healing wound, induced by high NEP, to an acute wound [wild-type (C57BL/6J) saline-treated mice] that follows the normal progression of healing, the chronic wound displayed delayed wound closure (day 7), significantly greater MMP-9 expression (day 0, 2 and 7) with no formation of granulation tissue, re-epithelization and angiogenesis (day 7). Proteomic analysis of the acute saline-treated and chronic high NEP wounds at day 2 indicated significant differences in expression of proteins such as Stefin-1, Stefin-3, Microtubule-associated protein 1B, Band 4.1-like protein 2, Caveolae-associated protein 1 and Gamma-synuclein. These proteins have not been previously described as involved in wound healing and may be directly involved or have downstream interactions in the wound healing processes. This study identified two proteins previously described as playing a role in wound healing, via their involvement in inflammation (Alpha-1- acid glycoprotein 1) and proliferation and remodelling (Nidogen-1). In conclusion, high NEP administration in the wound boarders of obese pre-diabetic mice resulted in a chronic wound model that better mimics human diabetic chronic wounds, which could be used to test various treatment strategies. High NEP administration resulted in less wound closure that could be explained by higher MMP-9, lower cytokine content in wound fluid and differences in several proteins identified by proteomic analysis.

AFRIKAANSE OPSOMMING: Chroniese wonde kom wêreldwyd onder miljoene mense met diabetes voor. Verskeie diermodelle vir diabetiese wondgenesing bestaan, maar geen repliseer tans chroniese wonde onder diabetiese pasiënte nie. Diermodelle wat nie voldoende met mensetoestande ooreenstem nie, belemmer die toepassing van belowende terapeutiese strategieë in kliniese omgewings. Substansie P is ʼn neuropeptied wat wondgenesing bevorder deur aan die neurokinien 1-reseptor te bind, wat die inflammatoriese proses in werking stel. Neutrale endopeptidase (NEP) is ʼn sinkmetalloprotease wat substansie P degradeer, waardeur die genesingsproses belemmer word. Verhoogde NEP ensimatiese aktiwiteit is sigbaar in chroniese wonde van diabetiese pasiënte, wat nie die geval in huidige diermodelle is nie. Die doel van hierdie studie was om die optimale dosis van NEP vir inspuiting in wondrande van vetsugtige prediabetiese muise (B6.Cg- Lepob/J, ob/ob) te bepaal ten einde ʼn chroniese niegenesende wond te skep. Daarna is die wondgenesingsdinamika van hierdie chroniese niegenesende wond met ʼn akute wond wat met ʼn soutoplossing behandel is, vergelyk. Benewens soutoplossingkontrole is drie konsentrasies van NEP (lae NEP [0.33 mg/μL], middelslag-NEP [0.68 mg/μL] of hoë NEP [1.02 mg/μL]) gebruik om ʼn dosisrepsonskromme te verkry. NEP-aktiwiteite het nie onder die behandelingsgroepe verskil nie, maar substansie P was wel laer in die wondareas vir die hoë NEP- vergeleke met die lae NEP-groepe. ʼn Kleiner hoeveelheid van substansie P was dus teenwoordig om wondgenesing met hoë NEP in werking te stel. Daar is spesifiek gevind dat MMP-9 hoër was in die hoë NEP- behandelde groep op dag 2, vergeleke met soutoplossing-, lae en middelslag-NEP-groepe. Eweneens was die sitokienprofiel in gemengde monsters van dag 2 se wondvloeistof die laagste met hoë NEP-behandeling. Hoë NEP-toepassing het dus wel enkele eienskappe van ʼn chroniese wond in die vroeë fases ná verwonding nageboots, insluitende vertraagde aanvang van inflammasie. Met vergelyking van die genesende dinamika van hierdie chroniese wond, deur hoë NEP geïnduseer, met ʼn akute wond (wilde tipe [C57BL/6J] soutoplossingbehandelde muise) het die chroniese wond vertraagde wondgenesing (dag 7) en aanmerklik groter MMP-9-uitdrukking (dag 0, 2 en 7) getoon, met geen vorming van granulasieweefsel, herepitelisasie en angiogenese in die wondarea van die chroniese wond nie (dag 7). Proteomiese analise van die akute soutoplossing- en chroniese hoë NEP-wonde op dag 2 het aanmerklike verskille in uitdrukking van proteïene (Stefin-1, Stefin-3, mikrotubulus-geassosieerde proteïen 1B, band 4.1-agtige proteïen 2, kaveola-geassosieerde proteïen 1 en gamma-sinukleïen) getoon, wat nog nie voorheen met betrekking tot wondgenesing beskryf is nie. Hierdie proteïene is dalk nie direk betrokke nie, maar het moontlike stroomaf-interaksies in die wondgenesingsproses. Hierdie studie het slegs twee proteïene geïdentifiseer wat volgens die literatuur ʼn rol in wondgenesing speel deur hul betrokkenheid by inflammasie (alfa-1-suur glikoproteïen 1) en proliferasie en hermodellering (Nidogen-1). Die gevolgtrekking is dat hoë NEP-toediening in die wondrande van vetsugtige prediabetiese muise ʼn wond geskep het wat ʼn chroniese wond beter naboots as laer NEP-konsentrasies of soutoplossingtoediening. Hoë NEP-toediening het tot minder wondgenesing gelei, wat die gevolg mag wees van hoër MMP-9, laer sitokien-inhoud in wondvog en verskille in verskeie proteïene wat deur proteomiese analise geïdentifiseer is.

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