George Tellides

Interferon-gamma elicits arteriosclerosis in the absence of leukocytes.

Atherosclerosis and post-transplant graft arteriosclerosis are both characterized by expansion of the arterial intima as a result of the infiltration of mononuclear leukocytes, the proliferation of vascular smooth muscle cells (VSMCs) and the accumulation of extracellular matrix. They are also associated with the presence of the immunomodulatory cytokine interferon-γ (IFN-γ). Moreover, in mouse models of atheroma formation or allogeneic transplantation, the serological neutralization or genetic absence of IFN-γ markedly reduces the extent of intimal expansion. However, other studies have found that exogenous IFN-γ inhibits cultured VSMC proliferation and matrix synthesis, and reduces intimal expansion in response to mechanical injury. This discrepancy is generally explained by the idea that IFN-γ either directly activates macrophages, or, by increasing antigen presentation, indirectly activates T cells within the lesions of atherosclerosis and graft arteriosclerosis. These activated leukocytes are thought to express the VSMC-activating cytokines and cell-surface molecules that cause the observed arteriosclerotic responses. Here we have inserted pig and human arteries into the aorta of immunodeficient mice, and we show that IFN-γ can induce arteriosclerotic changes in the absence of detectable immunocytes by acting on VSMCs to potentiate growth-factor-induced mitogenesis.

CD4+CD25+ regulatory T cells suppress allograft rejection mediated by memory CD8+ T cells via a CD30-dependent mechanism

CD4(+)CD25(+) regulatory T (Treg) cells suppress naive T cell responses, prevent autoimmunity, and delay allograft rejection. It is not known, however, whether Treg cells suppress allograft rejection mediated by memory T cells, as the latter mount faster and stronger immune responses than their naive counterparts. Here we show that antigen-induced, but not naive, Treg cells suppress allograft rejection mediated by memory CD8(+) T cells. Suppression was allospecific, as Treg cells induced by third-party antigens did not delay allograft rejection. In vivo and in vitro analyses revealed that the apoptosis of allospecific memory CD8(+) T cells is significantly increased in the presence of antigen-induced Treg cells, while their proliferation remains unaffected. Importantly, neither suppression of allograft rejection nor enhanced apoptosis of memory CD8(+) T cells was observed when Treg cells lacked CD30 or when CD30 ligand-CD30 interaction was blocked with anti-CD30 ligand Ab. This study therefore provides direct evidence that pathogenic memory T cells are amenable to suppression in an antigen-specific manner and identifies CD30 as a molecule that is critical for the regulation of memory T cell responses.

Small-diameter biodegradable scaffolds for functional vascular tissue engineering in the mouse model.

The development of neotissue in tissue engineered vascular grafts remains poorly understood. Advances in mouse genetic models have been highly informative in the study of vascular biology, but have been inaccessible to vascular tissue engineers due to technical limitations on the use of mouse recipients. To this end, we have developed a method for constructing sub-1mm internal diameter (ID) biodegradable scaffolds utilizing a dual cylinder chamber molding system and a hybrid polyester sealant scaled for use in a mouse model. Scaffolds constructed from either polyglycolic acid or poly-l-lactic acid nonwoven felts demonstrated sufficient porosity, biomechanical profile, and biocompatibility to function as vascular grafts. The scaffolds implanted as either inferior vena cava or aortic interposition grafts in SCID/bg mice demonstrated excellent patency without evidence of thromboembolic complications or aneurysm formation. A foreign body immune response was observed with marked macrophage infiltration and giant cell formation by post-operative week 3. Organized vascular neotissue, consisting of endothelialization, medial generation, and collagen deposition, was evident within the internal lumen of the scaffolds by post-operative week 6. These results present the ability to create sub-1mm ID biodegradable tubular scaffolds that are functional as vascular grafts, and provide an experimental approach for the study of vascular tissue engineering using mouse models.

Hyperplastic Cellular Remodeling of the Media in Ascending Thoracic Aortic Aneurysms

Background—Progressive medial degeneration and atrophy is thought to be a cause of ascending thoracic aortic aneurysms in the elderly. Extensive apoptosis of vascular smooth muscle cells (VSMCs) has been demonstrated in the media of abdominal aortic aneurysms. We investigated whether medial atrophy from loss of VSMCs occurs in primary ascending thoracic aortic aneurysms. Methods and Results—Morphometric analysis of 28 nonaneurysmal ascending thoracic aortas and 29 ascending thoracic aortic aneurysms was performed by directly measuring the thickness of their vascular layers and by indirectly calculating the area of their vascular compartments. The cellular and matrix composition of the media was assessed at the structural, protein, and transcript levels. Despite thinning of the media secondary to vascular dilatation, there was an overall increase in the medial area of aneurysms. VSMC density was preserved, implying cellular hyperplasia as a result of the increased medial mass. There was decreased expression of matrix proteins, despite sustained synthesis of these molecules, which was associated with evidence of increased matrix degradation. The remodeling and expansion of the media was most evident in comparisons between nonaneurysmal aortas versus smaller aneurysms and did not evolve further in larger aneurysms. Conclusions—The mechanisms for luminal enlargement in thoracic and abdominal aortic aneurysms differ significantly with regard to the survival of VSMCs and atrophy of the media but share common pathophysiology involving degeneration of the matrix. Hyperplastic cellular remodeling of the media in ascending thoracic aortic aneurysms may be an initial adaptive response to minimize increased wall stress resulting from vascular dilatation.

Tgfbr2 disruption in postnatal smooth muscle impairs aortic wall homeostasis

TGF-β is essential for vascular development; however, excess TGF-β signaling promotes thoracic aortic aneurysm and dissection in multiple disorders, including Marfan syndrome. Since the pathology of TGF-β overactivity manifests primarily within the arterial media, it is widely assumed that suppression of TGF-β signaling in vascular smooth muscle cells will ameliorate aortic disease. We tested this hypothesis by conditional inactivation of Tgfbr2, which encodes the TGF-β type II receptor, in smooth muscle cells of postweanling mice. Surprisingly, the thoracic aorta rapidly thickened, dilated, and dissected in these animals. Tgfbr2 disruption predictably decreased canonical Smad signaling, but unexpectedly increased MAPK signaling. Type II receptor-independent effects of TGF-β and pathological responses by nonrecombined smooth muscle cells were excluded by serologic neutralization. Aortic disease was caused by a perturbed contractile apparatus in medial cells and growth factor production by adventitial cells, both of which resulted in maladaptive paracrine interactions between the vessel wall compartments. Treatment with rapamycin restored a quiescent smooth muscle phenotype and prevented dissection. Tgfbr2 disruption in smooth muscle cells also accelerated aneurysm growth in a murine model of Marfan syndrome. Our data indicate that basal TGF-β signaling in smooth muscle promotes postnatal aortic wall homeostasis and impedes disease progression.

T cell–mediated vascular dysfunction of human allografts results from IFN-γ dysregulation of NO synthase

Allograft vascular dysfunction predisposes to arteriosclerosis and graft loss. We examined how dysfunction develops in transplanted human arteries in response to circulating allogeneic T cells in vivo using immunodeficient murine hosts. Within 7-9 days, transplanted arteries developed endothelial cell (EC) dysfunction but remained sensitive to exogenous NO. By 2 weeks, the grafts developed impaired contractility and desensitization to NO, both signs of VSMC dysfunction. These T cell-dependent changes correlated with loss of eNOS and expression of iNOS--the latter predominantly within infiltrating T cells. Neutralizing IFN-gamma completely prevented both vascular dysfunction and changes in NOS expression; neutralizing TNF reduced IFN-gamma production and partially prevented dysfunction. Inhibiting iNOS partially preserved responses to NO at 2 weeks and reduced graft intimal expansion after 4 weeks in vivo. In vitro, memory CD4+ T cells acted on allogeneic cultured ECs to reduce eNOS activity and expression of protein and mRNA. These effects required T cell activation by class II MHC antigens and costimulators (principally lymphocyte function-associated antigen-3, or LFA-3) on the ECs and were mediated by production of soluble mediators including IFN-gamma and TNF. We conclude that IFN-gamma is a central mediator of vascular dysfunction and, through dysregulation of NOS expression, links early dysfunction with late arteriosclerosis.

Interferon-γ Axis in Graft Arteriosclerosis

Cardiac transplantation is the most effective treatment for advanced heart failure. Despite improvements in immunosuppression therapy that prevent acute rejection, cardiac allografts fail at rates of 3% to 5% per posttransplant year. The hallmark morphological lesion of chronically failing cardiac allografts, also seen in chronic renal and liver graft failure, is luminal stenosis of blood vessels, especially of conduit arteries. Late graft failure results from widespread secondary ischemic injury to the graft parenchyma rather than direct immune-mediated damage. Although this process affects the entire graft vasculature, graft arteriosclerosis is a suitable term to describe the problem because it applies to different types of failing organs and because it emphasizes the central feature, namely an accelerated form of arterial injury and remodeling. The precise pathogenesis of graft arteriosclerosis is unknown. In this review, we make the case that the signature T-helper type 1 cytokine, interferon (IFN)-&ggr;, is a key effector in graft arteriosclerosis, which, together with the IFN-&ggr;–inducing cytokine interleukin-12 and IFN-&ggr;–inducible chemokines such as CXCR3 ligands, constitute a positive feedback loop for T-cell activation, differentiation, and recruitment that we refer to as the IFN-&ggr; axis. We evaluate the evidence to support this hypothesis in clinical observational and experimental animal studies. Additionally, we examine the regulation of IFN-&ggr; production within the artery wall, the effects of IFN-&ggr; on vessel wall cells, and the outcome of therapeutic agents on IFN-&ggr; production and signaling. These observations lead us to suggest that new therapies for graft arteriosclerosis should be optimized which focus on reducing IFN-&ggr; synthesis or actions.

Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells

Immune responses are suppressed in immunologically privileged sites, which may provide a unique opportunity to prolong allograft survival. However, it is unknown whether testicular immune privilege promotes transplantation tolerance. Mechanisms underlying immune privilege are also not well understood. Here we found that islet transplantation in the testis, an immunologically privileged site, generates much less memory CD8+ T cells but induces more Ag-specific CD4+CD25+ regulatory T cells than in a conventional site. These CD4+CD25+ cells exhibited the suppression of alloimmune responses in vivo and in vitro. Despite the immune regulation, intratesticular islet allografts all were rejected within 42 days after transplantation although they survived longer than renal subcapsular islet allografts. However, blocking CD40/CD40L costimulation induced the tolerance of intratesticular, but not renal subcapsular, islet allografts. Tolerance to intratesticular islet allografts spread to skin allografts in the non-privileged sites. Either transfer of memory CD8+ T cells or deletion of CD25+ T cells in vivo broke islet allograft tolerance. Thus, transplantation tolerance requires both costimulatory blockade, which suppresses acute allograft rejection, and a favorable balance between memory and regulatory T cells that could favorably prevent late allograft failure. These findings reveal novel mechanisms of immune privilege and provide direct evidence that testicular immune privilege fosters the induction of transplantation tolerance to allografts in both immunologically privileged and non-privileged sites.

Endothelial-to-mesenchymal transition drives atherosclerosis progression

The molecular mechanisms responsible for the development and progression of atherosclerotic lesions have not been fully established. Here, we investigated the role played by endothelial-to-mesenchymal transition (EndMT) and its key regulator FGF receptor 1 (FGFR1) in atherosclerosis. In cultured human endothelial cells, both inflammatory cytokines and oscillatory shear stress reduced endothelial FGFR1 expression and activated TGF-β signaling. We further explored the link between disrupted FGF endothelial signaling and progression of atherosclerosis by introducing endothelial-specific deletion of FGF receptor substrate 2 α (Frs2a) in atherosclerotic (Apoe(-/-)) mice. When placed on a high-fat diet, these double-knockout mice developed atherosclerosis at a much earlier time point compared with that their Apoe(-/-) counterparts, eventually demonstrating an 84% increase in total plaque burden. Moreover, these animals exhibited extensive development of EndMT, deposition of fibronectin, and increased neointima formation. Additionally, we conducted a molecular and morphometric examination of left main coronary arteries from 43 patients with various levels of coronary disease to assess the clinical relevance of these findings. The extent of coronary atherosclerosis in this patient set strongly correlated with loss of endothelial FGFR1 expression, activation of endothelial TGF-β signaling, and the extent of EndMT. These data demonstrate a link between loss of protective endothelial FGFR signaling, development of EndMT, and progression of atherosclerosis.

Role of Mechanotransduction in Vascular Biology Focus on Thoracic Aortic Aneurysms and Dissections

Thoracic aortic diseases that involve progressive enlargement, acute dissection, or rupture are influenced by the hemodynamic loads and mechanical properties of the wall. We have only limited understanding, however, of the mechanobiological processes that lead to these potentially lethal conditions. Homeostasis requires that intramural cells sense their local chemomechanical environment and establish, maintain, remodel, or repair the extracellular matrix to provide suitable compliance and yet sufficient strength. Proper sensing, in turn, necessitates both receptors that connect the extracellular matrix to intracellular actomyosin filaments and signaling molecules that transmit the related information to the nucleus. Thoracic aortic aneurysms and dissections are associated with poorly controlled hypertension and mutations in genes for extracellular matrix constituents, membrane receptors, contractile proteins, and associated signaling molecules. This grouping of factors suggests that these thoracic diseases result, in part, from dysfunctional mechanosensing and mechanoregulation of the extracellular matrix by the intramural cells, which leads to a compromised structural integrity of the wall. Thus, improved understanding of the mechanobiology of aortic cells could lead to new therapeutic strategies for thoracic aortic aneurysms and dissections.