Jonathan Choy

Increased ABCA1 activity protects against atherosclerosis.

The ABC transporter ABCA1 plays a key role in the first steps of the reverse cholesterol transport pathway by mediating lipid efflux from macrophages. Previously, it was demonstrated that human ABCA1 overexpression in vivo in transgenic mice results in a mild elevation of plasma HDL levels and increased efflux of cholesterol from macrophages. In this study, we determined the effect of overexpression of ABCA1 on atherosclerosis development. Human ABCA1 transgenic mice (BAC(+)) were crossed with ApoE(-/-) mice, a strain that spontaneously develop atherosclerotic lesions. BAC(+)ApoE(-/-) mice developed dramatically smaller, less-complex lesions as compared with their ApoE(-/-) counterparts. In addition, there was increased efflux of cholesterol from macrophages isolated from the BAC(+)ApoE(-/-) mice. Although the increase in plasma HDL cholesterol levels was small, HDL particles from BAC(+)ApoE(-/-) mice were significantly better acceptors of cholesterol. Lipid analysis of HDL particles from BAC(+)ApoE(-/-) mice revealed an increase in phospholipid levels, which was correlated significantly with their ability to enhance cholesterol efflux.

Granzyme B Induces Smooth Muscle Cell Apoptosis in the Absence of Perforin Involvement of Extracellular Matrix Degradation

Objective—T cell-induced cytotoxicity, of which granzyme B is a key mediator, is believed to contribute to the pathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of granzyme B-induced smooth muscle cell (SMC) death. Methods and Results—The addition of purified granzyme B alone to cultured SMCs caused a significant reduction in cell viability. Chromatin condensation, phosphatidylserine externalization, and membrane blebbing were observed, indicating that the mechanism of granzyme B-induced SMC death was through apoptosis. Activated splenocytes from perforin-knockout mice induced SMC death through a granzyme B-mediated pathway. Inhibition of the proteolytic activities of caspases and granzyme B prevented granzyme B-induced SMC death, whereas attenuation of granzyme B internalization with mannose-6-phosphate (M6P) did not. Further, granzyme B induced the cleavage of several SMC extracellular proteins, including fibronectin, and reduced focal adhesion kinase phosphorylation. Conclusions—These results indicate that granzyme B can induce apoptosis of SMCs in the absence of perforin by cleaving extracellular proteins, such as fibronectin.

Soluble Recombinant Coxsackievirus and Adenovirus Receptor Abrogates Coxsackievirus B3-Mediated Pancreatitis and Myocarditis in Mice

BACKGROUND Group B coxsackievirus infection can result in organ injury and inflammation. The coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF; CD55) have both been identified as receptors for coxsackievirus B3 (CVB3). We have shown elsewhere that early DAF-Fc treatment attenuates CVB3-induced myocarditis and virus replication. METHODS CAR was synthesized as a soluble IgG1-Fc fusion protein (CAR-Fc). In vitro, CAR-Fc blocked infection by 2 different strains of CVB3. A/J mice were infected in vivo with CVB3 and were administered CAR-Fc either 3 days before infection, during infection, or 3 days after infection and were compared with mice infected with virus alone and control animals. RESULTS All CAR-Fc treatment groups had reduced recoverable infectious virus in the heart. CAR-Fc treatment of mice, either preceding or concurrent with CVB3 infection, resulted in complete inhibition of myocardial lesion area, cell death and inflammation, and viral RNA. Early treatment also completely blocked inflammation and cell death in the pancreas, an organ that is normally very sensitive to infection. CONCLUSION To our knowledge, CAR-Fc is the only protein that has been shown to block coxsackievirus infection of the pancreas. However, regardless of the efficacy of the test protein, target tissue cannot be rescued after day 3 of infection in the A/J mouse model.

Coxsackievirus B3-Associated Myocardial Pathology and Viral Load Reduced by Recombinant Soluble Human Decay-Accelerating Factor in Mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). in vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.

Long-term effects of ovariectomy and estrogen replacement treatment on endothelial function in mature rats

OBJECTIVES Estrogen replacement therapy (ERT) improves blood flow through various mechanisms including an augmented release of nitric oxide (NO). We report on the long-term effects of estrogen loss on vascular function and endothelial regulation. METHODS Male, female, ovariectomized and ovariectomized+ERT treated rats were used. Female rats were ovariectomized at 12 weeks of age and received ERT via subcutaneously implanted 90-day release pellets. Vasodilation to acetylcholine (ACh) was studied in tail artery segments; arterial blood was collected for measurements of 17-beta-estradiol and stable metabolites of NO (nitrate/nitrite). Some arterial segments were harvested for TUNEL staining to determine endothelial apoptosis. RESULTS Ovariectomy caused a rapid loss of estradiol that was negated by ERT. Likewise, there was also a loss in plasma NO. Loss of ACh-mediated dilations were age-dependent and were significant in males and untreated ovariectomized rats, with the change being maximal after 12 weeks of ovariectomy. After 12 weeks post-ovariectomy, there were no time dependent changes in ACh sensitivity in either group. Dilations to ACh were maintained in females and age-matched ERT ovariectomized rats over time. TUNEL staining of the endothelium (at 6 months of age) revealed apoptotic changes with the rank order male>ovariectomized>female, or ERT treated ovariectomized female rats. CONCLUSIONS In a rat model of surgical menopause, loss of endothelial function is maximal 12 weeks after ovariectomy. Apoptosis of endothelial cells is greatest in arteries from male rats. Our data suggests that early ERT treatment may be an important consideration for reducing endothelium-dependent vascular dysfunction.

Cyclosporine treatment preserves coronary resistance artery function in rat cardiac allografts.

BACKGROUND A marked decline in vascular myogenic response occurs during the course of rat cardiac allograft rejection. Two important contributory features are an inducible nitrous oxide synthase (iNOS)-catalyzed, NO-mediated vasodilation and a loss of smooth muscle function. In this study, we examine the effect of cyclosporine immunosuppressive therapy on the alleviation of arterial dysfunction of coronary resistance arteries in allografts using pressure myography. METHODS Rats receiving heterotopic abdominal cardiac transplantation were treated with cyclosporine (5 mg/kg), Cremophore or distilled water. Coronary septal arteries (internal diameter 200 microm) were dissected from isograft (Lewis to Lewis) and allograft (Fisher to Lewis) rat hearts at Day 21 post-transplantation and mounted on a pressure myograph. Pressure-induced vasoconstriction was measured before and after iNOS inhibition with aminoguanidine (AG; 100 micromol/liter). Both endothelium-based (ACh-induced) and endothelium-independent (sodium nitroprusside-induced) vasorelaxation were also recorded in each group. RESULTS Pressure-induced myogenic contraction was reduced in allograft coronary arteries at Day 21 post-transplantation compared with matched isografts (p < 0.05). AG potentiated myogenic tone in allograft arteries, but had no effect on untreated Day 21 isograft vessels, indicating the presence of iNOS-based relaxation only in allograft vessels. Depolarization-induced vasoconstriction was lower in allograft compared with isograft arteries (p < 0.05). Cyclosporine therapy also improved depolarization-induced constriction in allograft vessels compared with untreated groups (p < 0.05). Furthermore, cyclosporine therapy preserved endothelium-based and endothelium-independent vasorelaxation in allograft arteries at Day 21 post-transplantation. CONCLUSIONS Cyclosporine immunosuppressive therapy has a significant effect on the alleviation of early endothelial and smooth muscle dysfunction in coronary allograft arteries.

Bim Regulates Alloimmune-Mediated Vascular Injury Through Effects on T-Cell Activation and Death

Objective—Bim is a proapoptotic Bcl-2 protein known to downregulate immune responses and to also be required for antigen-induced T-cell activation. However, it is not known how the effect of Bim on these offsetting processes determines the outcome of allogeneic immune responses. We have defined the role of Bim in regulating alloantigen-driven T-cell responses in a model of vascular rejection. Approach and Results—Bim was required for proliferation of CD4 and CD8 T cells, and for interleukin-2 production, in T cells stimulated with alloantigen in vitro. Moreover, a partial reduction in Bim expression was sufficient to attenuate T-cell activation, whereas a complete elimination of Bim was required to prevent CD4 T-cell death in response to cytokine withdrawl. When alloimmune-mediated vascular rejection was examined using an aortic interposition model, there was significantly less intimal thickening in Bim+/−, but not Bim−/−, graft recipients. T-cell proliferation in response to allograft arteries was significantly reduced in both Bim+/− and Bim−/− mice, but cell death was attenuated only in Bim−/− animals. Conclusions—Bim controls both T-cell activation and death in response to alloantigen stimulation. These processes act cooperatively to determine the outcome of immune responses in allograft arteries.

Mouse model of alloimmune-induced vascular rejection and transplant arteriosclerosis.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

Life and Death Signaling Pathways in CVB3-Induced Myocarditis

When Rodbell and co-workers discovered the role of cyclic adenosine monophosphate (cAMP) as a cellular second messenger, the world of life sciences moved to a new conceptual plateau.1–5 Since then, signaling, in and out of cells, through or bypassing the genome has become a protracted and revealing focus of investigation and discovery. In 1979, the breakthrough discovery that the activity of an enzyme can be reversibly regulated through the action of phosphatases and kinases greatly enriched the concept of cellular homeostasis: how it is maintained in health and disrupted in disease states.6 Tyrosine phosphorylation was also uncovered as a mediator of growth control,7 and mitogen-activated protein kinase signaling was discovered by groups studying transcription factor phosphorylation and protein-serine kinases activated by receptor protein tyrosine kinases .8 The identification of a large family of small monomeric G proteins was a giant step forward in understanding intracellular signaling.9 Ras was the first protein found to be conserved from yeast to vertebrates, and Ras-associated signaling pathways include Sos,10 guanosine triphosphateguanosine diphosphate (GTP-GDP) exchange, Ras-GAP, and Raf.11 Finally, the discovery of transcription factor NFKB was important in understanding nuclear translocation of a cytoplasmically sequestered transcription factor.12