Acidic pH and acidic enzymes in atherosclerosis

Atherosclerosis is an inflammatory disease characterized by accumulation of lipids and fibrous connective tissue in the arterial wall. Recently, it has been suggested that decrease in the pH of extracellular fluid of the arterial intima may enhance LDL accumulation by increasing binding of the LDL to matrix proteoglycans and also by making the plaque more favorable for acidic enzymes to be active. Many lysosomal acidic enzymes have been found in atherosclerotic plaques. In this thesis, we were able to induce secretion of lysosomal acidic cathepsin F from human monocyte-derived macrophages by stimulation with angiotensin II. We also showed that LDL pre-proteolyzed with cathepsin S was more prone to subsequent hydrolytic modifications by lipases. Especially acidic secretory sphingomyelinase was able to hydrolyze pre-proteolyzed LDL even at neutral pH. We also showed that the proteolyzed and lipolyzed LDL particles were able to bind more efficiently to human aortic proteoglycans. In addition, the role of extracellular acidic pH on the ability of macrophages to internalize LDL was studied. At acidic pH, the production of cell surface proteoglycans in macrophages was increased as well as the binding of native and modified LDL to cell surface proteoglycans. Furthermore, macrophages cultured at acidic pH showed increased internalization of modified and native LDL leading to foam cell formation. This thesis revealed various mechanisms by which acidic pH can increase LDL retention and accumulation in the arterial intima and has the potential to increase the progression of atherosclerosis.Ateroskleroosi on valtimon seinämän tulehdussairaus. Sen merkittävimmät ilmenemismuodot ovat sydäninfarkti ja aivoinfarkti. Ateroskleroosissa veren kolesterolia kuljettavat LDL-hiukkaset tunkeutuvat valtimoiden seinämään, jonka seurauksena kehittyy ateroskleroottinen plakki. Plakin kasvaminen ahtauttaa valtimon onteloa ja heikentää veren virtausta. Plakki voi myös revetä, jolloin muodostuu paikallinen verihyytymä, joka voi tukkia valtimon ja aiheuttaa hapen puutteessa olevaan kudokseen infarktin eli kuolion. Viimeaikaisissa tutkimuksissa paksuntuneesta valtimon seinämästä on löydetty alueita, joissa solunulkoisen tilan pH on voimakkaasti laskenut, paikallisesti jopa alle pH kuuden. Happamassa LDL-hiukkasten on todettu sitoutuvan voimakkaammin seinämän proteoglykaaneihin, mikä edesauttaa LDL-hiukkasten kertymistä seinämään, sekä altistaa LDL-hiukkaset seinämässä sijaitsevien entsyymien aiheuttamalle muuntumiselle. Tässä väitöskirjassa on tutkittu happamuuden vaikutusta LDL-hiukkasten kertymiseen ateroskleroottisessa plakissa esiintyviin makrofageihin. Lisäksi väitöskirjassa tutkittiin happamien entsyymien eritystä makrofageista sekä niiden vaikutusta solunulkoisessa tilassa. Tutkimuksemme osoittivat verisuonen seinämästä löytyvän angiotensiiini II:n pystyvän aiheuttamaan happaman katepsiini F-entsyymin erittymisen solunulkoiseen tilaan. Lisäksi katepsiini S:n todettiin muuntavan LDL-hiukkaset alttiimmaksi solunukoisten lipaasien vaikutukselle, minkä seurauksena muun muassa LDL-hiukkasten sitoutuminen soluväliaineen proteoglykaaneihin vahvistui. Happamassa makrofagit tuottivat enemmän solun pinnan proteoglykaaneita ja LDL-hiukkasten sitoutumisen näihin todettiin lisääntyvän. Tämän seurauksena muuntuneiden ja jopa muuntumattomien LDL-hiukkasten sisäänotto makrofageihin lisääntyi happamassa pH:ssa. Hapan pH lisää LDL-hiukkasten tarttumista valtimon seinämästä eristettyihin rakenteisiin ja kiihdyttää ateroskleroosille tyypillisten kolesterolitäytteisten makrofagien muodostumista. Niinpä nämä valtimon seinämän happamat alueet voivat toimia ateroskleroosin kehittymistä voimakkaasti edistävinä keskuksina

Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding

Background and aims: Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. Methods: Mature human MCs were differentiated from human peripheral blood-derived CD34(+) progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. Results: Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. Conclusions: The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A2 (sPLA2-IIa and sPLA2-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA2-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA2-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA2-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis