Fergus Davison

Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice

Recent evidence indicates that vascular progenitor cells may be the source of smooth muscle cells (SMCs) that accumulate in atherosclerotic lesions, but the origin of these progenitor cells is unknown. To explore the possibility of vascular progenitor cells existing in adults, a variety of tissues from ApoE-deficient mice were extensively examined. Immunohistochemical staining revealed that the adventitia in aortic roots harbored large numbers of cells having stem cell markers, e.g., Sca-1(+) (21%), c-kit(+) (9%), CD34(+) (15%), and Flk1(+) cells (4%), but not SSEA-1(+) embryonic stem cells. Explanted cultures of adventitial tissues using stem cell medium displayed a heterogeneous outgrowth, for example, islands of round-shaped cells surrounded by fibroblast-like cell monolayers. Isolated Sca-1(+) cells were able to differentiate into SMCs in response to PDGF-BB stimulation in vitro. When Sca-1(+) cells carrying the LacZ gene were transferred to the adventitial side of vein grafts in ApoE-deficient mice, beta-gal(+) cells were found in atherosclerotic lesions of the intima, and these cells enhanced the development of the lesions. Thus, a large population of vascular progenitor cells existing in the adventitia can differentiate into SMCs that contribute to atherosclerosis. Our findings indicate that ex vivo expansion of these progenitor cells may have implications for cellular, genetic, and tissue engineering approaches to vascular disease.

Endothelial Replacement and Angiogenesis in Arteriosclerotic Lesions of Allografts Are Contributed by Circulating Progenitor Cells

Background—Endothelial regeneration and angiogenesis in the intima of the arterial wall are key events in the pathogenesis of transplantation arteriosclerosis. The traditional hypothesis that damaged endothelial cells are replaced by remaining cells of the donor vessel has been challenged by recent observations, but the cell origins of large arteries and microvessels are still not well established. Methods and Results—Aortic segments were allografted between Balb/c and TIE2-LacZ (C57BL/6) mice expressing &bgr;-galactosidase (gal) in endothelial cells. &bgr;-gal+ cells in TIE2-LacZ vessels grafted to Balb/c mice completely disappeared, whereas the positive cells found in Balb/c aorta allografted into TIE2-LacZ mice 4 weeks after surgery indicated a host origin. En face analysis of allograft vessels displayed a unique distribution of &bgr;-gal+ cells on the surface at 3 days, 1 week, and 4 weeks. Interestingly, 35±19% &bgr;-gal+ cells were found in arterial segments allografted into chimeric mice with TIE2-LacZ bone marrows. Furthermore, endothelial cells of microvessels within allografts had a &bgr;-gal+ staining in the media at 1 week and in the neointimal lesions and adventitia at 4 weeks. Allograft studies in chimeric mice demonstrated that &bgr;-gal+ cells of microvessels in transplant arteriosclerosis were derived from bone marrow progenitors. Conclusions—We provide strong evidence that endothelial cells of neointimal lesions in allografts are derived from circulating progenitor cells and that bone marrow–derived progenitors are responsible for angiogenesis of the allograft, that is, the formation of microvessels in transplant arteriosclerosis.

Smooth Muscle Cells in Transplant Atherosclerotic Lesions Are Originated From Recipients, but Not Bone Marrow Progenitor Cells

Background—Smooth muscle cell (SMC) accumulation in the intima of vessels is a key event in the pathogenesis of transplant atherosclerosis. The traditional hypothesis that SMCs in the lesion are derived from the media of the donor vessel has been challenged by recent observations, but the cell origin is still not well established. Methods and Results—Here, we use a simplified model of artery allografts in transgenic mice to clearly identify the source of SMCs in transplant atherosclerosis. Aortic segments donated by BALB/c mice allografted to ROSA26 (C57B/6) mice expressing &bgr;-galactosidase (gal) in all tissues showed that neointimal cells derived exclusively from host cells. It was also demonstrated that SMCs of neointimal and atherosclerotic lesions in vessels allografted to mice expressing &bgr;-gal only in SMCs (SM-LacZ) or to apoE-deficient/SM-LacZ mice originated from the recipient, and not donor vessels. Interestingly, bone marrow transplantation of SM-LacZ &bgr;-gal–expressing cells into aortic allograft recipients revealed completely negative &bgr;-gal staining of neointimal and atherosclerotic lesions. However, a population of &bgr;-gal–positive cells in lesions of allografts was observed in chimeric mice with ROSA26 &bgr;-gal–expressing marrow cells. When bone marrow cells from both ROSA26 and SM-LacZ mice were cultured and stimulated with platelet-derived growth factor-BB, &agr;-actin and &bgr;-gal double-positive cells were found, suggesting that bone marrow cells have an ability to differentiate into SMCs. Conclusions—Thus, we provide strong evidence that SMCs of neointimal and atherosclerotic lesions in allografts are derived from the recipients and that non–bone marrow–derived progenitor cells are a possible source of SMCs in atherosclerotic lesions.

Circulating progenitor cells regenerate endothelium of vein graft atherosclerosis, which is diminished in ApoE-deficient mice.

Abstract— Previously we showed that a large number of endothelial cells in vein grafts undergo apoptosis or necrosis during the first few days followed by endothelial regeneration. In the present study, we investigated endothelial cell death and regeneration in vein grafts using transgenic mice carrying LacZ genes driven by an endothelial TIE2 promoter. When a vein fragment from TIE2-LacZ was isografted into the carotid artery of wild-type mice, the number of &bgr;-gal+ cells were reduced at 3 days and disappeared completely by 4 weeks after grafting. Conversely, &bgr;-gal+ cells were observed on the surface of vein segments donated by wild-type mice isografted into TIE2-LacZ mice at 1 week and reached confluence by 4 weeks, suggesting recipient origins of endothelial cells. Interestingly, &bgr;-gal+ cells were evenly distributed on the surface of the whole vein segment grafted into TIE2-LacZ mice, indicating a contribution of circulating progenitor cells. When wild-type veins were grafted into a chimeric mouse carrying TIE2-LacZ genes in bone marrow cells, a proportion of cells displayed a &bgr;-gal+ staining. Furthermore, the number of CD34+ and Flk+ progenitor cells in blood of apoE-deficient mice were significantly lower than those of wild-type controls, which coincided with diminished &bgr;-gal+ endothelial cells on the surface of vein grafts in TIE2-LacZ/apoE−/− mice. Thus, we provide the first evidence that endothelial cells of vein grafts are derived from circulating progenitor cells, of which one-third are derived from bone marrow progenitor cells. Hyperlipidemia due to apoE deficiency results in a lower number of endothelial progenitors in blood and correlated with enhanced atherosclerosis. The full text of this article is available online at http://www.circresaha.org.

Both Donor and Recipient Origins of Smooth Muscle Cells in Vein Graft Atherosclerotic Lesions

Abstract— Smooth muscle cell (SMC) accumulation in the inner layer of the vessel wall is a key event in the pathogenesis of atherosclerosis in vein grafts, but the origin of the cells in these lesions has yet to be shown. Herein, we use animal models of vein grafts in transgenic mice to clearly identify the sources of SMCs in atherosclerosis. Vena cava segments were isografted to carotid arteries between four types of transgenic mice, including SM-LacZ expressing &bgr;-galactosidase (&bgr;-gal) in vascular SMCs, SM-LacZ/apoE−/−, ROSA26 expressing &bgr;-gal in all tissues, and wild-type mice. &bgr;-gal–positive cells were observed in neointimal and atherosclerotic lesions of all vein segments grafted between LacZ transgenic and wild-type mice. Double staining for &bgr;-gal and cell nuclei revealed that about 40% of SMCs originated from hosts and 60% from the donor vessel. This was confirmed by double labeling of the Y-chromosome and &agr;-actin in the lesions of sex-mismatched vein grafts. The possibility that bone marrow cells were the source of SMCs in grafts was eliminated by the absence of &bgr;-gal staining in atherosclerotic lesions of chimeric mice. Furthermore, vein SMCs of SM-LacZ mice did not express &bgr;-gal in situ, but did so when these cells appeared in atherosclerotic lesions in vivo, suggesting that hemodynamic forces may be crucial for SMC differentiation. Thus, we provide the first evidence of SMC origins in the atherosclerotic lesions of vein grafts, which will be essential for providing insight into new types of therapy for the disease. The full text of this article is available at http://www.circresaha.org.

A new mutation of the cardiac troponin T gene causing familial hypertrophic cardiomyopathy without left ventricular hypertrophy

AIM To screen for a mutation of the cardiac troponin T gene in two families where there had been sudden deaths without an increase in left ventricular mass but with myocardial disarray suggesting hypertrophic cardiomyopathy. METHODS DNA from affected individuals from both families was used to screen the cardiac troponin T gene on an exon by exon basis. Mutation screening was achieved by polymerase chain reaction and direct sequencing. Where appropriate, a mutation was confirmed by restriction digest. RESULTS A novel missense mutation of exon 9 was found in the affected individuals of one of the families. This mutation at amino acid 94 resulted in the substitution of arginine for leucine and was not found in 100 normal control samples. A mutation of the cardiac troponin T gene was excluded in the second family. CONCLUSIONS A mutation of the gene for the sarcomeric protein cardiac troponin T can cause familial hypertrophic cardiomyopathy with marked myocyte disarray and frequent premature sudden death in the absence of myocardial hypertrophy at clinical or macroscopic level.

Cytosine methylation confers instability on the cardiac troponin T gene in hypertrophic cardiomyopathy

Editor—Hypertrophic cardiomyopathy (HCM) is an inherited disease (MIM 192600, 115195) of the heart muscle, characterised by unexplained left ventricular hypertrophy. HCM is also one of the major causes of sudden cardiac death,1sometimes occurring in young asymptomatic people.2-4Although sporadic forms do rarely occur,5 generally HCM has an autosomal dominant pattern of inheritance caused by mutations of the genes coding for proteins of the cardiac sarcomere. Subjects with HCM caused by mutations in the cardiac troponin T ( cTNT ) gene have been clinically shown to be at increased risk of sudden death,6 which may occur even in the absence of marked morphological abnormalities.7Since incomplete penetrance of the clinical phenotype, measured by ECG and echocardiographic parameters, is one of the hallmarks of “troponin” disease, the identification of cTNT mutation in probands would facilitate identification of “at risk” relatives who may not fulfil clinical diagnostic criteria. In the course of a study undertaken to characterise the cTNT mutation profile in HCM patients, we identified a cluster of mutations in exons 8 and 9. Five out of the 11 mutations published to date in this gene have been found in exons 88 and 9.7-10 We report here a novel Arg94Cys de novo mutation in a female patient presenting with HCM bringing the total of cTNT mutations to 12. Four of the mutations found in exon 9, Arg92Trp, Arg92Gln, Arg94Cys, and Ala104Val, involve C→T transitions (or G→A transitions in the opposite strand) within CpG dinucleotides. Approximately 70% of the cytosines within CpG dinucleotides in the mammalian genome contain highly mutable 5-methyl-cytosine (5mC) residues. These residues are not randomly distributed and the majority of the genome is CpG depleted.11 Although some CpG dinucleotides are found within coding regions, most CpG residues are in CpG islands, …

Histopathological features of sudden death in cardiac troponin T disease

Background—Patients with hypertrophic cardiomyopathy (HCM) are at increased risk of premature death; this is particularly apparent for patients with mutations of the troponin T gene. Myocyte disarray and interstitial fibrosis, pathological features of HCM, may be determinants in these deaths. The relation between genotype, pathological phenotype, and mode of death has not been explored. Methods and Results—Seventy-five hearts with HCM were examined. DNA was available in 50 for screening of the troponin T gene. The macroscopic findings, percentage of disarray, percentage of fibrosis, and percentage of small-vessel disease were correlated with the genotype. A troponin T mutation was identified in 9 of the 50 patients, 8 of whom died suddenly. Patients with a troponin T mutation were younger (mean age, 21.0 years [range, 6 to 37] versus 39.1 years [range, 14 to 72];P <0.0001), had more sudden death (P =0.02), and had lower heart weights, less fibrosis, and greater disarray than other HCM patients (mean heart weight, 380.3±105.4 versus 585.0±245.7 g, P =0.002; mean fibrosis, 0.7±0.4% versus 2.6±2.8%, P =0.001; mean disarray, 46.2±7.2% versus 24.1±15.9%, P <0.0001; and mean small-vessel disease, 11.7±14.6 versus 14.1±8.7, P =0.6, respectively). Similarly, patients with troponin T mutations who died suddenly had lower heart weights and greater disarray than patients who died suddenly with unknown genotype (ie, troponin T mutation excluded) (mean heart weight, 429.8±75.4 versus 559.6±204.43 g, P =0.04, and mean disarray, 40.1±9.4% versus 20.2±12.6%, P =0.002, respectively). Conclusions—Patients with troponin T mutations had severe disarray, with only mild hypertrophy and fibrosis. These patients died suddenly and at an especially early age. We propose that extensive myocyte disarray in the absence of marked hypertrophy is the pathological substrate for sudden death in these patients.