| 1. |
- Karlöf, Eva, et al.
(författare)
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Correlation of computed tomography with carotid plaque transcriptomes associates calcification with lesion-stabilization
- 2019
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Ingår i: Atherosclerosis. - Stockholm : ELSEVIER IRELAND LTD. - 0021-9150 .- 1879-1484. ; 288, s. 175-185
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Tidskriftsartikel (refereegranskat)abstract
- Background and aims: Unstable carotid atherosclerosis causes stroke, but methods to identify patients and lesions at risk are lacking. We recently found enrichment of genes associated with calcification in carotid plaques from asymptomatic patients. Here, we hypothesized that calcification represents a stabilising feature of plaques and investigated how macro-calcification, as estimated by computed tomography (CT), correlates with gene expression profiles in lesions. Methods: Plaque calcification was measured in pre-operative CT angiographies. Plaques were sorted into high- and low-calcified, profiled with microarrays, followed by bioinformatic analyses. Immunohistochemistry and qPCR were performed to evaluate the findings in plaques and arteries with medial calcification from chronic kidney disease patients. Results: Smooth muscle cell (SMC) markers were upregulated in high-calcified plaques and calcified plaques from symptomatic patients, whereas macrophage markers were downregulated. The most enriched processes in high-calcified plaques were related to SMCs and extracellular matrix (ECM) organization, while inflammation, lipid transport and chemokine signaling were repressed. These findings were confirmed in arteries with high medial calcification. Proteoglycan 4 (PRG4) was identified as the most upregulated gene in association with plaque calcification and found in the ECM, SMA+ and CD68+/TRAP + cells. Conclusions: Macro-calcification in carotid lesions correlated with a transcriptional profile typical for stable plaques, with altered SMC phenotype and ECM composition and repressed inflammation. PRG4, previously not described in atherosclerosis, was enriched in the calcified ECM and localized to activated macrophages and smooth muscle-like cells. This study strengthens the notion that assessment of calcification may aid evaluation of plaque phenotype and stroke risk.
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| 2. |
- Röhl, Samuel, et al.
(författare)
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Transcriptomic profiling of experimental arterial injury reveals new mechanisms and temporal dynamics in vascular healing response
- 2020
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Ingår i: JVS-Vascular Science. - : Elsevier BV. - 2666-3503. ; 315, s. E14-E14
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Endovascular interventions cause arterial injury and induce a healing response to restore vessel wall homeostasis. Complications of defective or excessive healing are common and result in increased morbidity and repeated interventions. Experimental models of intimal hyperplasia are vital for understanding the vascular healing mechanisms and resolving the clinical problems of restenosis, vein graft stenosis, and dialysis access failure. Our aim was to systematically investigate the transcriptional, histologic, and systemic reaction to vascular injury during a prolonged time. Methods: Balloon injury of the left common carotid artery was performed in male rats. Animals (n = 69) were euthanized before or after injury, either directly or after 2 hours, 20 hours, 2 days, 5 days, 2 weeks, 6 weeks, and 12 weeks. Both injured and contralateral arteries were subjected to microarray profiling, followed by bioinformatic exploration, histologic characterization of the biopsy specimens, and plasma lipid analyses. Results: Immune activation and coagulation were key mechanisms in the early response, followed by cytokine release, tissue remodeling, and smooth muscle cell modulation several days after injury, with reacquisition of contractile features in later phases. Novel pathways related to clonal expansion, inflammatory transformation, and chondro-osteogenic differentiation were identified and immunolocalized to neointimal smooth muscle cells. Analysis of uninjured arteries revealed a systemic component of the reaction after local injury, underlined by altered endothelial signaling, changes in overall tissue bioenergy metabolism, and plasma high-density lipoprotein levels. Conclusions: We demonstrate that vascular injury induces dynamic transcriptional landscape and metabolic changes identifiable as early, intermediate, and late response phases, reaching homeostasis after several weeks. This study provides a temporal “roadmap” of vascular healing as a publicly available resource for the research community.
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| 3. |
- Seime, Till, et al.
(författare)
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Biomechanical Assessment of Macro-Calcification in Human Carotid Atherosclerosis and Its Impact on Smooth Muscle Cell Phenotype
- 2022
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 11:20, s. 3279-
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Tidskriftsartikel (refereegranskat)abstract
- Intimal calcification and vascular stiffening are predominant features of end-stage atherosclerosis. However, their role in atherosclerotic plaque instability and how the extent and spatial distribution of calcification influence plaque biology remain unclear. We recently showed that extensive macro calcification can be a stabilizing feature of late-stage human lesions, associated with a reacquisition of more differentiated properties of plaque smooth muscle cells (SMCs) and extracellular matrix (ECM) remodeling. Here, we hypothesized that biomechanical forces related to macro-calcification within plaques influence SMC phenotype and contribute to plaque stabilization. We generated a finite element modeling (FEM) pipeline to assess plaque tissue stretch based on image analysis of preoperative computed tomography angiography (CTA) of carotid atherosclerotic plaques to visualize calcification and soft tissues (lipids and extracellular matrix) within the lesions. Biomechanical stretch was significantly reduced in tissues in close proximity to macro calcification, while increased levels were observed within distant soft tissues. Applying this data to an in vitro stretch model on primary vascular SMCs revealed upregulation of typical markers for differentiated SMCs and contractility under low stretch conditions but also impeded SMC alignment. In contrast, high stretch conditions in combination with calcifying conditions induced SMC apoptosis. Our findings suggest that the load bearing capacities of macro calcifications influence SMC differentiation and survival and contribute to atherosclerotic plaque stabilization.
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| 4. |
- Seime, Till
(författare)
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The role of smooth muscle cells in calcification of atherosclerotic plaques
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Calcification (CALC) is a predominant feature of late-stage cardiovascular disease (CVD) but responsible mechanisms and its contribution to the risk of clinical events remain unclear. Formation of highly mineralized extracellular matrix (ECM) leads to progressive aortic valve stenosis (AVS) and has been identified as a surrogate marker for atherosclerotic disease burden. However, we previously found enrichment of genes associated with CALC in atherosclerotic carotid lesions from asymptomatic patients and in patients on statin therapy. This thesis aimed to investigate how CALC correlates with gene expression profiles in human specimens of carotid plaques as well as AVS and functionally characterize the underlying mechanisms associated to osteogenic phenotypic transformation of structural cells. Study I explored gene expression profiles and biological pathways related to macro-CALC in human carotid lesions, estimated by computed tomography (CT). Microarray profiling, bioinformatic analysis and histological validation based on high- vs low-CALC plaques revealed upregulation of smooth muscle cell (SMC) markers in high-CALC plaques, whereas macrophage markers were downregulated. The most enriched processes in high-CALC plaques were related to SMC differentiation and ECM organization, while inflammation, lipid transport and chemokine signaling were repressed. Proteoglycan 4 (PRG4) was identified as the most upregulated gene in association with plaque CALC and found in the ECM overlapping with SMA, CD68 and tartrate-resistant acid phosphatase (TRAP) positive cells. Study II characterized PRG4 in the context of AVS and aortic valve CALC. Transcriptomic, histological and immunohistochemical (IHC) analysis of human aortic valves from patients undergoing aortic valve replacement showed significant upregulation of PRG4 in thickened and CALC regions of aortic valves compared with healthy regions. In addition, PRG4 positively associated with mRNA expression of proteins involved in cardiovascular CALC. Treatment of human valve interstitial cells (VICs) with recombinant human PRG4 (rhPRG4) enhanced phosphate (Pi) induced CALC and increased bone morphogenetic protein 2 (BMP2) expression. Study III analyzed the role of PRG4 in vascular remodeling and intimal CALC. PRG4, detected by IHC, localized to SMCs in early human intimal thickening, while in advanced lesions it was found in the ECM, surrounding macro-CALC. In vivo mouse and rat models showed increased Prg4 expression in SMCs during intimal hyperplasia, correlating with osteogenic markers and transforming growth factor b (Tgfb). Moreover, PRG4 was enriched around intimal plaque CALC in apolipoprotein E deficient mice (ApoE-/-) on warfarin. In vitro, PRG4 was induced in primary human vascular SMCs by TGFb and calcifying conditions, while SMC markers were repressed. Silencing experiments showed that PRG4 expression was driven by transcription factors mothers against decapentaplegic homolog 3 (SMAD3) and SRY-box transcription factor 9 (SOX9). The addition of rhPRG4 increased ectopic SMC calcification, while arresting cell migration, proliferation and osteogenic transformation. Study IV assessed the influence of biomechanical forces, related to atherosclerotic carotid macro-CALC, on SMC phenotype and plaque stability. Finite element modeling (FEM), based on preoperative CT images, identified that biomechanical stretch was significantly reduced in close proximity to macro-CALC, while pathologically increased levels were observed within distant soft tissues. In vitro modeling of these conditions revealed upregulation of markers for SMC differentiation and contractility under low stretch but also impeded SMC alignment and increased osteogenic transdifferentiation. In contrast, high-stretch in combination with calcifying conditions induced rapid SMC apoptosis, suggesting a contribution to atherosclerotic plaque stabilization by the load-bearing capacities of macro-CALC. Overall, this thesis demonstrates that vascular disease pathology related to CALC can be comprehensively described by linking clinical diagnostics and underlying biological processes via in silico analysis, thereby contributing to the basic understanding of disease progression and patient specific phenotypic variability.
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| 5. |
- Skenteris, Nikolaos T, et al.
(författare)
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Osteomodulin attenuates smooth muscle cell osteogenic transition in vascular calcification
- 2022
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Ingår i: Clinical and Translational Medicine. - : Wiley. - 2001-1326. ; 12:2, s. 1-22
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Tidskriftsartikel (refereegranskat)abstract
- RATIONALE: Vascular calcification is a prominent feature of late-stage diabetes, renal and cardiovascular disease (CVD), and has been linked to adverse events. Recent studies in patients reported that plasma levels of osteomodulin (OMD), a proteoglycan involved in bone mineralisation, associate with diabetes and CVD. We hypothesised that OMD could be implicated in these diseases via vascular calcification as a common underlying factor and aimed to investigate its role in this context.METHODS AND RESULTS: In patients with chronic kidney disease, plasma OMD levels correlated with markers of inflammation and bone turnover, with the protein present in calcified arterial media. Plasma OMD also associated with cardiac calcification and the protein was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, circulating OMD was increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data showed that OMD was upregulated in atherosclerotic compared to control arteries, particularly in calcified plaques, where OMD expression correlated positively with markers of smooth muscle cells (SMCs), osteoblasts and glycoproteins. Immunostaining confirmed that OMD was abundantly present in calcified plaques, localised to extracellular matrix and regions rich in α-SMA+ cells. In vivo, OMD was enriched in SMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE-/- mice on warfarin. In vitro experiments revealed that OMD mRNA was upregulated in SMCs stimulated with IFNγ, BMP2, TGFβ1, phosphate and β-glycerophosphate, and by administration of recombinant human OMD protein (rhOMD). Mechanistically, addition of rhOMD repressed the calcification process of SMCs treated with phosphate by maintaining their contractile phenotype along with enriched matrix organisation, thereby attenuating SMC osteoblastic transformation. Mechanistically, the role of OMD is exerted likely through its link with SMAD3 and TGFB1 signalling, and interplay with BMP2 in vascular tissues.CONCLUSION: We report a consistent association of both circulating and tissue OMD levels with cardiovascular calcification, highlighting the potential of OMD as a clinical biomarker. OMD was localised in medial and intimal α-SMA+ regions of calcified cardiovascular tissues, induced by pro-inflammatory and pro-osteogenic stimuli, while the presence of OMD in extracellular environment attenuated SMC calcification.
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