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Background: Aim of this study was to investigate immune cells and subsets in different stages of human coronary artery disease with a novel multiplex immunohistochemistry (mIHC) technique. Methods: Human left anterior descending coronary artery specimens were analyzed: eccentric intimal thickening (N = 11), pathological intimal thickening (N = 10), fibroatheroma (N = 9), and fibrous plaque (N = 9). Eccentric intimal thickening was considered normal, and pathological intimal thickening, fibroatheroma, and fibrous plaque were considered diseased coronary arteries. Two mIHC panels, consisting of six and five primary antibodies, autofluoresence, and DAPI, were used to detect adaptive and innate immune cells. Via semi-automated analysis, (sub)types of immune cells in whole plaques and specific plaque regions were quantified. Results: Increased numbers of CD3+ T cells (P < 0.001), CD20+ B cells (P = 0.013), CD68+ macrophages (P = 0.003), CD15+ neutrophils (P = 0.017), and CD31+ endothelial cells (P = 0.024) were identified in intimas of diseased coronary arteries compared to normal. Subset analyses of T cells and macrophages showed that diseased coronary arteries contained an abundance of CD3+CD8- non-cytotoxic T cells and CD68+CD206- non-M2-like macrophages. Proportions of CD3+CD45RO+ memory T cells were similar to normal coronary arteries. Among pathological intimal thickening, fibroatheroma, and fibrous plaque, all immune cell numbers and subsets were similar. Conclusions: The type of immune response does not differ substantially between different stages of plaque development and may provide context for mechanistic research into immune cell function in atherosclerosis. We provide the first comprehensive map of immune cell subtypes across plaque types in coronary arteries demonstrating the potential of mIHC for vascular research., Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

Background and aims: Randomized clinical studies have shown a reduction in cardiovascular outcomes with glucagon-like peptide 1 receptor agonist (GLP-1RA) treatment with the hypothesized mechanisms being an underlying effect on atherosclerosis. Here, we aimed to assess the pharmacological effects of semaglutide in an atheroprone murine model that recapitulates central mechanisms related to vascular smooth muscle cell (VSMC) phenotypic switching and endothelial dysfunction known to operate within the atherosclerotic plaque. Methods: In study A, we employed an electrical current to the carotid artery in ApoE−/− mice to induce severe VSMC injury and death, after which the arteries were allowed to heal for 4 weeks. In study B, a constrictive cuff was added for 6 h at the site of the healed segment to induce a disturbance in blood flow. Results: Compared to vehicle, semaglutide treatment reduced the intimal and medial area by ∼66% (p = 0.007) and ∼11% (p = 0.0002), respectively. Following cuff placement, expression of the pro-inflammatory marker osteopontin and macrophage marker Mac-2 was reduced (p < 0.05) in the semaglutide-treated group compared to vehicle. GLP-1R were not expressed in murine carotid artery and human coronary vessels with and without atherosclerotic plaques, and semaglutide treatment did not affect proliferation of cultured primary human VSMCs. Conclusions: Semaglutide treatment reduced vessel remodelling following electrical injury and blood flow perturbation in an atheroprone mouse model. This effect appears to be driven by anti-inflammatory and -proliferative mechanisms independent of GLP-1 receptor-mediated signalling in the resident vascular cells. This mechanism of action may be important for cardiovascular protection.

Background and aims: Randomized clinical studies have shown a reduction in cardiovascular outcomes with glucagon-like peptide 1 receptor agonist (GLP-1RA) treatment with the hypothesized mechanisms being an underlying effect on atherosclerosis. Here, we aimed to assess the pharmacological effects of semaglutide in an atheroprone murine model that recapitulates central mechanisms related to vascular smooth muscle cell (VSMC) phenotypic switching and endothelial dysfunction known to operate within the atherosclerotic plaque. Methods: In study A, we employed an electrical current to the carotid artery in ApoE−/− mice to induce severe VSMC injury and death, after which the arteries were allowed to heal for 4 weeks. In study B, a constrictive cuff was added for 6 h at the site of the healed segment to induce a disturbance in blood flow. Results: Compared to vehicle, semaglutide treatment reduced the intimal and medial area by ∼66% (p = 0.007) and ∼11% (p = 0.0002), respectively. Following cuff placement, expression of the pro-inflammatory marker osteopontin and macrophage marker Mac-2 was reduced (p < 0.05) in the semaglutide-treated group compared to vehicle. GLP-1R were not expressed in murine carotid artery and human coronary vessels with and without atherosclerotic plaques, and semaglutide treatment did not affect proliferation of cultured primary human VSMCs. Conclusions: Semaglutide treatment reduced vessel remodelling following electrical injury and blood flow perturbation in an atheroprone mouse model. This effect appears to be driven by anti-inflammatory and -proliferative mechanisms independent of GLP-1 receptor-mediated signalling in the resident vascular cells. This mechanism of action may be important for cardiovascular protection.

Background Immunization with oxidized LDL (oxLDL) reduces atherosclerosis in rodents. We tested the hypothesis that treatment with a human recombinant monoclonal antibody against oxLDL will reduce the burden or composition of atherosclerotic lesions in hypercholesterolemic minipigs. Methods and results Thirty-eight hypercholesterolemic minipigs with defective LDL receptors were injected with an oxLDL antibody or placebo weekly for 12 weeks. An 18F-fluorodeoxyglucose positron emission tomography (FDG PET) scan (n = 9) was performed before inclusion and after 3 months of treatment. Blood samples were obtained prior to each injection. Following the last injection all animals were sacrificed, and the heart, aorta, and iliac arteries were removed. The left anterior descending coronary artery was sectioned at 5 mm intervals for quantitative and qualitative assessments of atherosclerosis, including immunohistochemical phenotyping of macrophages using a pan-macrophage marker (CD68) and markers for putative pro-atherogenic (cathepsin S) and atheroprotective (CD163) macrophages. Aorta, right coronary artery, and left iliac artery were stained en face with Sudan IV and the amount of atherosclerosis quantified. There was no effect of treatment on plasma lipid profile, vascular FDG-PET signal or the amount of atherosclerosis in any of the examined arteries. However, immunostaining of coronary lesions revealed reduced cathepsin S positivity in the treated group compared with placebo (4.8% versus 8.2% of intima area, p = 0.03) with no difference in CD68 or CD163 positivity. Conclusions In hypercholesterolemic minipigs, treatment with a human recombinant monoclonal antibody against oxLDL reduced cathepsin S in coronary lesions without any effect on the burden of atherosclerosis or aortic FDG-PET signal.

RATIONALE: Atherosclerosis can be achieved in animals by germline genetic engineering, leading to hypercholesterolemia, but such models are constrained to few species and strains, and they are difficult to combine with other powerful techniques involving genetic manipulation or variation.OBJECTIVE: To develop a method for induction of atherosclerosis without germline genetic engineering.METHODS AND RESULTS: Recombinant adeno-associated viral vectors were engineered to encode gain-of-function proprotein convertase subtilisin/kexin type 9 mutants, and mice were given a single intravenous vector injection followed by high-fat diet feeding. Plasma proprotein convertase subtilisin/kexin type 9 and total cholesterol increased rapidly and were maintained at high levels, and after 12 weeks, mice had atherosclerotic lesions in the aorta. Histology of the aortic root showed progression of lesions to the fibroatheromatous stage. To demonstrate the applicability of this method for rapid analysis of the atherosclerosis susceptibility of a mouse strain and for providing temporal control over disease induction, we demonstrated the accelerated atherosclerosis of mature diabetic Akita mice. Furthermore, the versatility of this approach for creating atherosclerosis models also in nonmurine species was demonstrated by inducing hypercholesterolemia and early atherosclerosis in Golden Syrian hamsters.CONCLUSIONS: Single injections of proprotein convertase subtilisin/kexin type 9-encoding recombinant adeno-associated viral vectors are a rapid and versatile method to induce atherosclerosis in animals. This method should prove useful for experiments that are high-throughput or involve genetic techniques, strains, or species that do not combine well with current genetically engineered models.

RATIONALE: Atherosclerosis can be achieved in animals by germline genetic engineering, leading to hypercholesterolemia, but such models are constrained to few species and strains, and they are difficult to combine with other powerful techniques involving genetic manipulation or variation.OBJECTIVE: To develop a method for induction of atherosclerosis without germline genetic engineering.METHODS AND RESULTS: Recombinant adeno-associated viral vectors were engineered to encode gain-of-function proprotein convertase subtilisin/kexin type 9 mutants, and mice were given a single intravenous vector injection followed by high-fat diet feeding. Plasma proprotein convertase subtilisin/kexin type 9 and total cholesterol increased rapidly and were maintained at high levels, and after 12 weeks, mice had atherosclerotic lesions in the aorta. Histology of the aortic root showed progression of lesions to the fibroatheromatous stage. To demonstrate the applicability of this method for rapid analysis of the atherosclerosis susceptibility of a mouse strain and for providing temporal control over disease induction, we demonstrated the accelerated atherosclerosis of mature diabetic Akita mice. Furthermore, the versatility of this approach for creating atherosclerosis models also in nonmurine species was demonstrated by inducing hypercholesterolemia and early atherosclerosis in Golden Syrian hamsters.CONCLUSIONS: Single injections of proprotein convertase subtilisin/kexin type 9-encoding recombinant adeno-associated viral vectors are a rapid and versatile method to induce atherosclerosis in animals. This method should prove useful for experiments that are high-throughput or involve genetic techniques, strains, or species that do not combine well with current genetically engineered models.

Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.