Aggravating effects of extracellular matrix in vascular stiffening, atherosclerosis and hypertension - a translational study

Kimmie Bjerre Christensen

doi:10.21996/vd8p-yj71

Aggravating effects of extracellular matrix in vascular stiffening, atherosclerosis and hypertension - a translational study

Bidragets oversatte titel: Forværrende mekanismer af ekstracellulær matrix på vaskulær stivhed, åreforkalkning og hypertension - et translationelt studie

Kimmie Bjerre Christensen

Publikation: Afhandling › Ph.d.-afhandling

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Abstract

Hovedformålet med denne Ph.D. er at karakterisere mikrofibrillar-associeret protein 4 (MFAP4)s rolle i kardiovaskulære sygdomme. For at undersøge dette, har vi brugt MFAP4-deficiente mus (Mfap4-/- ) og antiMFAP4 antistofbehandling for at undersøge de MFAP4-afhængige mekanismer i udviklingen af åreforkalkning, hypertension og aldersrelateret vaskulær stivhed (ADVS). Derudover har vi i et translationelt studie undersøgt surfaktant protein-D (SP-D)s egnethed som prognostisk seromarkør i prækliniske patienter med perifær arteriesygdom (PAD).

Formålet i manuskript I var at visualisere ekspressionen af MFAP4 i humane carotid arterier med åreforkalkningslæsioner vha. in situ hybridisering (ISH) samt undersøge hvilke celler som udtrykker MFAP4 i væv fra humane koronar arterier med åreforkalkningslæsioner vha. single cell ribonucleic acid sekventering (scRNAseq). Derudover ville vi karakterisere udviklingen af åreforkalkning i ApoE-/- Mfap4+/+og ApoE-/- Mfap4- /- mus efter høj-fedt diæt (HFD) i 24 uger. ApoE-/- Mfap4-/- musene var beskyttede mod udvikling af åreforkalkningslæsioner, med et signifikant mindre areal dækket af læsioner i aorta. Disse mus viste også at have mere stabile læsioner med mindre nekrose, øget kollagen deponering og mindre angiogenese. Ydermere viste ApoE-/- Mfap4-/- musene sig at have en mindre andel af CD36-udtrykkende celler i
åreforkalkningslæsionerne, som også blev bakket op af in vitro forsøg der viste, at MFAP4 direkte øger genekspressionen af CD36 i primære humane makrofager. For at verificere effekten af MFAP4 på udviklingen af åreforkalkning, udførte vi et behandlingsstudie af ApoE- /- Mfap4+/+ mus indgivet to ugentlige intraperitoneale injektioner med anti-MFAP4 antistof eller isotype kontrol i de sidste 10 uger af HFD. Dette forsøg bekræftede at MFAP4 øger udviklingen af åreforkalkning, da
anti-MFAP4 behandling resulterede i et mindre areal dækket af åreforkalkningslæsioner i aorta samt mindre
inflammation.

Formålet med manuskript II var at undersøge de MFAP4-afhængige mekanismer i en angiotensin (Ang)IIinduceret hypertensionsmodel samt i aldersrelateret vaskulær stivhed i mus. Mfap4+/+ og Mfap4-/- mus fik indsat en mini-osmotisk pumpe subkutant, som udskilte AngII eller saltvand i 21 dage for at udvikle hypertensive eller normotensive (kontrol) mus. Kontinuerlig blodtryksmåling i vågne hypertensive mus viste
at Mfap4-/- mus havde et signifikant lavere blodtryk end Mfap4+/+ mus. Blodtryksresultaterne kunne ikke forklares ud fra strukturelle ændringer i hverken konduktive eller resistensarterier. Måling af karstivhed vha. wire og trykmyografi viste at hypertensive Mfap4-/- mus havde mindre stive resistensarterier end hypertensive Mfap4+/+ mus, som delvist kunne forklare det lavere blodtryk. Derudover fandt vi at
mesenteriekar fra hypertensive Mfap4-/- mus havde en signifikant højere protein-ekspression af endotel nitrogen oxid syntase (eNOS) end Mfap4+/+ mus, som indikerede at Mfap4-/- mus havde mere afslappede mesenteriske resistensarterier. Vi fandt ingen MFAP4-aflængig effekt i udvikling af ADVS i unge (6-7 måneder) og gamle (21-23 måneder) mus.

Formålet med manuskript III var at undersøge potentialet for plasma SP-D som uafhængig prædiktor af dødelighed (uafhængig af årsag), kardiovaskulære hændelser (major adverse cardiovascular events, MACE), kardiovaskulære hændelser som påvirker lemmerne (major adverse limb events, MALE) eller en kombination (MACLE) i prækliniske PAD patienter. Vi fandt ingen association mellem høj plasma SP-D (³200ng/mL) og
øget risiko for MACE, MALE eller MACLE. Dog kunne vi bekræfte at SP-D fungerer som en uafhængig prædiktor af dødelighed, som også er fundet i andre studier.

Manuskript IV indeholder et review som beskriver den dualistiske rolle som SP-D har i kardiovaskulære sygdomme. Her beskrives det, at SP-D både har pro- og anti-inflammatoriske funktioner i udviklingen af åreforkalkning og at denne mekanisme er afhængig af multimeriseringen af SP-D. Således har trimer og
monomer SP-D en mere pro-inflammatorisk rolle hvorimod dodekamer SP-D har anti-inflammatoriske egenskaber.

Bidragets oversatte titel	Forværrende mekanismer af ekstracellulær matrix på vaskulær stivhed, åreforkalkning og hypertension - et translationelt studie
Originalsprog	Engelsk
Bevilgende institution	Syddansk Universitet
Vejledere/rådgivere	Sørensen, Grith Lykke, Hovedvejleder Schlosser, Anders Grønnegaard, Bivejleder Bloksgaard, Maria, Bivejleder
Dato for forsvar	18. mar. 2022
Udgiver	Syddansk Universitet. Det Sundhedsvidenskabelige Fakultet
DOI	https://doi.org/10.21996/vd8p-yj71
Status	Udgivet - 2. mar. 2022

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10.21996/vd8p-yj71

Open Access VersionForlagets udgivne version, 89 MB

1 Tidsskriftartikel

The Dual Role of Surfactant Protein-D in Vascular Inflammation and Development of Cardiovascular Disease
Colmorten, K. B., Nexoe, A. B. & Sorensen, G. L., 20. sep. 2019, I: Frontiers in Immunology. 10, 12 s., 2264.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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abstract = "The main purpose of this Ph.D. thesis is to characterize the role of microfibrillar-associated protein 4 (MFAP4) in cardiovascular pathologies. We used MFAP4-deficient mice (Mfap4-/-) and anti-MFAP4 antibody treatment to examine MFAP4- dependent mechanisms in atherosclerosis, hypertension and age-dependent vascular stiffness. Moreover, a translational study of surfactant protein-D (SP-D) and its ability to function as a prognosticseromarker in preclinical peripheral artery disease (PAD) patients, was performed. In manuscript I, we aimed to visualize MFAP4 expression in human carotid atherosclerotic lesions by in situ hybridization (ISH) and examine MFAP4 expressing cell clusters in human coronary atherosclerotic lesions by single cell ribonucleic acid sequencing (scRNAseq). Furthermore, we wanted to characterize the atherosclerotic lesion development in ApoE-/- Mfap4+/+ and ApoE-/- Mfap4-/- mice fed a high fat diet (HFD) for 24 weeks. We showed that ApoE-/- Mfap4-/- mice were protected from atherosclerotic lesion development, with decreased aortic lesion area. Lesions from ApoE-/- Mfap4-/- mice showed a more stable phenotype with less intralesional necrosis, increased collagen deposition and decreased angiogenesis. Moreover, ApoE-/-Mfap4-/- mice presented with a decreased proportion of cluster of differentiation (CD)36 expressing cells within atherosclerotic lesions and in vitro studies on primary human macrophages showed that MFAP4 directly increased CD36 gene expression. To verify MFAP4-dependent effects on atherosclerosis development, we performed an independent study with ApoE-/- Mfap4+/+ mice injected intraperitoneally with anti-MFAP4 antibody or isotype control. Mice were injected twice a week for the last ten weeks of HFD to assess the therapeutic potential of MFAP4 inhibition. The causal effects of MFAP4 on atherosclerosisdevelopment were confirmed, with anti-MFAP4 antibody treatment successfully attenuating atherosclerotic lesion area and inflammation.In manuscript II, we aimed to characterize the MFAP4-dependent effects in an angiotensin (Ang)II-induced hypertensive mouse model and examine MFAP4-dependent effects on age-dependent vascular stiffness (ADVS). Mfap4+/+ and Mfap4-/- mice had a mini-osmotic pump inserted subcutaneously, infusing AngII orsaline for 21 days to develop hypertensive and normotensive control mice, respectively. Blood pressure (BP) measurements in conscious mice showed a significant decreased BP in AngII-infused Mfap4-/- mice compared to AngII-infused Mfap4+/+ littermates. The BP results could not be explained by differences in structural properties of resistance or conductance arteries.Stiffness of aorta, carotid and mesenteric arteries were assessed by wire or pressure myography and showed a decreased stiffness of mesenteric resistance arteries in hypertensive Mfap4-/- mice, which in part could explain the decreased BP. Moreover, we found a significant increased protein expression of endothelialnitrogen oxide synthase (eNOS) in mesenteric arteries, implying that hypertensive Mfap4-/- mice had more relaxed mesenteric resistance arteries. We found no MFAP4-dependent effects on ADVS in young (6-7 months) and aged (21-23 months) mice. In manuscript III, we aimed to examine the ability of SP-D to function as an independent predictor of major adverse cardiovascular events (MACE), major adverse limb events (MALE), combined MACE and MALE (MACLE) and all-cause mortality in preclinical PAD patients. We found no association between high plasma SP-D (³200ng/mL) and increased risk of MACE, MALE or MACLE. However, we confirmed that SP-D is an independent predictor of all-cause mortality, as found in other studies. In manuscript IV, we did a review describing the dual role of SP-D in cardiovascular diseases (CVDs). Here we describe that SP-D has both pro- and anti-inflammatory roles in development of atherosclerosis and that this mechanism is highly dependent on the multimerization of SP-D. Trimeric and monomeric SP-D show proinflammatory properties whereas dodecameric SP-D shows anti-inflammatory properties. ",

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AB - The main purpose of this Ph.D. thesis is to characterize the role of microfibrillar-associated protein 4 (MFAP4) in cardiovascular pathologies. We used MFAP4-deficient mice (Mfap4-/-) and anti-MFAP4 antibody treatment to examine MFAP4- dependent mechanisms in atherosclerosis, hypertension and age-dependent vascular stiffness. Moreover, a translational study of surfactant protein-D (SP-D) and its ability to function as a prognosticseromarker in preclinical peripheral artery disease (PAD) patients, was performed. In manuscript I, we aimed to visualize MFAP4 expression in human carotid atherosclerotic lesions by in situ hybridization (ISH) and examine MFAP4 expressing cell clusters in human coronary atherosclerotic lesions by single cell ribonucleic acid sequencing (scRNAseq). Furthermore, we wanted to characterize the atherosclerotic lesion development in ApoE-/- Mfap4+/+ and ApoE-/- Mfap4-/- mice fed a high fat diet (HFD) for 24 weeks. We showed that ApoE-/- Mfap4-/- mice were protected from atherosclerotic lesion development, with decreased aortic lesion area. Lesions from ApoE-/- Mfap4-/- mice showed a more stable phenotype with less intralesional necrosis, increased collagen deposition and decreased angiogenesis. Moreover, ApoE-/-Mfap4-/- mice presented with a decreased proportion of cluster of differentiation (CD)36 expressing cells within atherosclerotic lesions and in vitro studies on primary human macrophages showed that MFAP4 directly increased CD36 gene expression. To verify MFAP4-dependent effects on atherosclerosis development, we performed an independent study with ApoE-/- Mfap4+/+ mice injected intraperitoneally with anti-MFAP4 antibody or isotype control. Mice were injected twice a week for the last ten weeks of HFD to assess the therapeutic potential of MFAP4 inhibition. The causal effects of MFAP4 on atherosclerosisdevelopment were confirmed, with anti-MFAP4 antibody treatment successfully attenuating atherosclerotic lesion area and inflammation.In manuscript II, we aimed to characterize the MFAP4-dependent effects in an angiotensin (Ang)II-induced hypertensive mouse model and examine MFAP4-dependent effects on age-dependent vascular stiffness (ADVS). Mfap4+/+ and Mfap4-/- mice had a mini-osmotic pump inserted subcutaneously, infusing AngII orsaline for 21 days to develop hypertensive and normotensive control mice, respectively. Blood pressure (BP) measurements in conscious mice showed a significant decreased BP in AngII-infused Mfap4-/- mice compared to AngII-infused Mfap4+/+ littermates. The BP results could not be explained by differences in structural properties of resistance or conductance arteries.Stiffness of aorta, carotid and mesenteric arteries were assessed by wire or pressure myography and showed a decreased stiffness of mesenteric resistance arteries in hypertensive Mfap4-/- mice, which in part could explain the decreased BP. Moreover, we found a significant increased protein expression of endothelialnitrogen oxide synthase (eNOS) in mesenteric arteries, implying that hypertensive Mfap4-/- mice had more relaxed mesenteric resistance arteries. We found no MFAP4-dependent effects on ADVS in young (6-7 months) and aged (21-23 months) mice. In manuscript III, we aimed to examine the ability of SP-D to function as an independent predictor of major adverse cardiovascular events (MACE), major adverse limb events (MALE), combined MACE and MALE (MACLE) and all-cause mortality in preclinical PAD patients. We found no association between high plasma SP-D (³200ng/mL) and increased risk of MACE, MALE or MACLE. However, we confirmed that SP-D is an independent predictor of all-cause mortality, as found in other studies. In manuscript IV, we did a review describing the dual role of SP-D in cardiovascular diseases (CVDs). Here we describe that SP-D has both pro- and anti-inflammatory roles in development of atherosclerosis and that this mechanism is highly dependent on the multimerization of SP-D. Trimeric and monomeric SP-D show proinflammatory properties whereas dodecameric SP-D shows anti-inflammatory properties.

U2 - 10.21996/vd8p-yj71

DO - 10.21996/vd8p-yj71

M3 - Ph.D. thesis

PB - Syddansk Universitet. Det Sundhedsvidenskabelige Fakultet

ER -

Aggravating effects of extracellular matrix in vascular stiffening, atherosclerosis and hypertension - a translational study

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The Dual Role of Surfactant Protein-D in Vascular Inflammation and Development of Cardiovascular Disease

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