Jian-Hua Qiao

Molecular Imaging of Cardiac Cell Transplantation in Living Animals Using Optical Bioluminescence and Positron Emission Tomography

Background—The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques. Methods and Results—For optical bioluminescence charged-coupled device imaging, rats (n=20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3×106 to 5×105) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3×106 cells: day 1 (627 000±15%), day 2 (346 100±21%), day 4 (112 800±20%), day 8 (78 860±24%), day 12 (67 780±12%), and day 16 (62 200±5% p · s−1 · cm2−1 · sr−1). For micro–positron emission tomography imaging, rats (n=20) received cardiomyoblasts (3×106) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48±0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01±0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays. Conclusions—The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.

Role of Group II Secretory Phospholipase A2 in Atherosclerosis 1. Increased Atherogenesis and Altered Lipoproteins in Transgenic Mice Expressing Group IIa Phospholipase A2

Some observations have suggested that the extracellular group IIa phospholipase A2 (sPLA2), previously implicated in chronic inflammatory conditions such as arthritis, may contribute to atherosclerosis. We have examined this hypothesis by studying transgenic mice expressing the human enzyme. Compared with nontransgenic littermates, the transgenic mice exhibited dramatically increased atherosclerotic lesions when maintained on a high-fat, high-cholesterol diet. Surprisingly, the transgenic mice also exhibited significant atherosclerotic lesions when maintained on a low-fat chow diet. Immunohistochemical staining indicated that sPLA2 was present in the atherosclerotic lesions of the transgenic mice. On both chow and atherogenic diets, the transgenic mice exhibited decreased levels of HDLs and slightly increased levels of LDLs compared with nontransgenic littermates. These data indicate that group IIa sPLA2 may promote atherogenesis, in part, through its effects on lipoprotein levels. These data also provide a possible mechanism for the observation that there is an increased incidence of coronary artery disease in many chronic inflammatory diseases.

Pathology of atheromatous lesions in inbred and genetically engineered mice. Genetic determination of arterial calcification.

We report comprehensive pathological studies of atheromatous lesions in various inbred mouse strains fed a high-fat, high-cholesterol diet and in two genetically engineered strains that develop spontaneous lesions on a low-fat chow diet. Coronary and aortic lesions were studied with respect to anatomic locations, lesion severity, calcification, and lipofuscin deposition. Surprisingly, the genetic determinants for coronary fatty lesion formation differed in part from those for aortic lesion development. This suggests the existence of genetic factors acting locally as well as systematically in lesion development. We used immunohistochemical analyses to determine the cellular and molecular compositions of the lesions. The aortic lesions contained monocyte/macrophages, lipid, apolipoprotein B, serum amyloid A proteins, and immunoglobulin M and showed expression of vascular cell adhesion molecule-1 and tumor necrosis factor-alpha, all absent in normal arteries. In certain strains, advanced lesions developed in which smooth muscle cells were commonly observed. The lesions in mice targeted for a null mutation in the apolipoprotein E gene were much larger, more widely dispersed, and more fibrous, cellular, and calcified in nature than the lesions in laboratory inbred strains. When apolipoprotein A-II transgenic mice were maintained on a low-fat chow diet, the lesions in these mice were relatively small and located in the very proximal regions of the aorta. There were clear differences in the occurrence of arterial wall calcification among genetically distinct inbred mouse strains, indicating for the first time a genetic component in this clinically significant trait. Analysis of a genetic cross indicated a complex pattern of calcification inheritance with incomplete penetrance.

Role of group II secretory phospholipase A2 in atherosclerosis. 2. Potential involvement of biologically active oxidized phospholipids

Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization-mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL.

Role for Sterol Regulatory Element-Binding Protein in Activation of Endothelial Cells by Phospholipid Oxidation Products

Oxidized phospholipids, including oxidation products of palmitoyl-arachidonyl-phosphatidyl choline (PAPC), are mediators of inflammation in endothelial cells (ECs) and known to induce several chemokines, including interleukin-8 (IL-8). In this study, we show that oxidized PAPC (OxPAPC), which accumulates in atherosclerotic lesions, paradoxically depletes endothelial cholesterol, causing caveolin-1 internalization from the plasma membrane to the endoplasmic reticulum and Golgi, and activates sterol regulatory element-binding protein (SREBP). Cholesterol loading reversed these effects. SREBP activation resulted in increased transcription of the low-density lipoprotein receptor, a target gene of SREBP. We also provide evidence that cholesterol depletion and SREBP activation are signals for OxPAPC induction of IL-8. Cholesterol depletion by methyl-&bgr;-cyclodextrin induced IL-8 synthesis in a dose-dependent manner. Furthermore, cholesterol loading of ECs by either the cholesterol–cyclodextrin complex or caveolin-1 overexpression inhibited OxPAPC induction of IL-8. These observations suggest that changes in cholesterol level can modulate IL-8 synthesis in ECs. The OxPAPC induction of IL-8 was mediated through the increased binding of SREBP to the IL-8 promoter region, as revealed by mobility shift assays. Overexpression of either dominant-negative SREBP cleavage-activating protein or 25-hydroxycholesterol significantly suppressed the effect of OxPAPC on IL-8 transcription. A role for SREBP activation in atherosclerosis is suggested by the observation that EC nuclei showed strong SREBP staining in human atherosclerotic lesions. The current studies suggest a novel role for endothelial cholesterol depletion and subsequent SREBP activation in inflammatory processes in which phospholipid oxidation products accumulate.

Immunoperoxidase staining for C4d on paraffin-embedded tissue in cardiac allograft endomyocardial biopsies: comparison to frozen tissue immunofluorescence.

C4d deposition in microvasculature is a marker for humoral rejection. The authors compared a recently developed C4d immunoperoxidase (IP) method for paraffin-embedded tissue to immunofluorescence (IF) of frozen tissue. Of 315 frozen endomyocardial biopsies with IF staining for C4d, 280 were negative and 35 were positive. Negative controls were 17 negative biopsies and 11 biopsies with myocyte necrosis. The extent of IP and IF staining was graded as 0 to 3+. Staining intensity and the number and type of positive vessels were recorded. Staining patterns in Quilty lesions (QL) and foci of acute cellular rejection (ACR) were also evaluated. In 34 biopsies with sufficient tissue, IP criteria of 2+/3+, or more than 10 to 20 positive vessels per 10 high-power fields detected 25.0% (1/4), 18.2% (2/11), and 84.2% (16/19) of 1+, 2+, and 3+ IF-positive biopsies, respectively, without false positives. Considering C4d IF 3+ as positive resulted in 84.2% (16/19) sensitivity and 93.0% specificity (40/43). Intensely stained capillaries predominated in six of seven biopsies when more than 100 capillaries per 10 high-power fields were positive. Seventy percent (7/10) of IP 2+ and 3+ biopsies showed positive capillaries in QLs, while 36.4% (4/11) of IP 1+ and negative biopsies did. All eight IP 2+/3+ biopsies showed positive capillaries in ACR foci, while 25.0% (1/4) of IP-negative biopsies did. Capillary staining in QLs and areas of ACR reflects overall C4d deposition. In conclusion, IP staining of 2+/3+ is highly sensitive and specific for C4d positivity. The authors recommend considering 2+ and 3+ as positive staining when using the IP technique.

Genetic Determination of Cartilaginous Metaplasia in Mouse Aorta

Calcification frequently occurs in atherosclerotic plaques in humans, but the cellular and genetic factors contributing to this pathological trait are unknown. We previously reported that the arterial calcification among inbred strains is genetically determined, and we now report that cartilaginous metaplasia, associated with the presence of arterial chondrocytes that express type II collagen, may underlie this calcification. Both uncalcified and calcified cartilaginous metaplasia were often colocalized with aortic atheromatous lesions and calcification, and clear genetic differences were observed in the occurrence of aortic cartilaginous metaplasia among inbred strains. Analysis of a genetic cross between strains C57BL/6J (exhibiting aortic cartilaginous metaplasia) and C3H/HeJ (no aortic cartilaginous metaplasia) revealed a recessive inheritance pattern; thus, F1 mice were entirely devoid of cartilaginous metaplasia, in common with the C3H/HeJ parental strain. Analyses of an F2 cross and a set of recombinant inbred strains derived from parental strains C57BL/6J and C3H/HeJ were consistent with a major gene effect exhibiting incomplete penetrance. The occurrence of aortic calcification was correlated with the occurrence of cartilaginous metaplasia in these genetic crosses, suggesting a link between the traits. Finally, we observed widespread calcified cartilaginous metaplasia within spontaneous atherosclerotic lesions in mice targeted for a null mutation in the apoE gene, suggesting that cartilaginous metaplasia is a potential pathway for artery wall calcification associated with the atherosclerotic plaque.

Genetic loci for diet-induced atherosclerotic lesions and plasma lipids in mice.

Genetic factors independent of those affecting plasma lipid levels are a major contributor to risk for atherosclerosis in humans, yet the basis for these is poorly understood. This study examined plasma lipids and diet-induced atherosclerosis in 16-month-old female mice of strains C56BL/6J and DBA/2J. Mice of the parental strains, from recombinant inbred strains derived from these (BXD RI), and F2 progeny were fed an atherogenic diet for 16 weeks, beginning at 1 year of age. This induced atherosclerotic lesion formation in both parental strains, accompanied by increased plasma LDL levels. However, individual BXD RI strains and the BXD F2 mice demonstrated a range of atherosclerotic lesion formation that was not or at best weakly correlated with plasma lipid levels. Quantitative trait locus (QTL) analysis of the BXD F2 mice identified a locus with significant linkage (lod 4.5) for aortic lesion size on Chromosome (Chr) 10 that was independent of plasma lipids. Other loci with suggestive or significant linkage for various plasma lipid measures were identified on Chr 2, 3, 4, 5, 6, 7, 11, and 17. In this intercross, the genes primarily influencing atherosclerosis are distinct from those controlling plasma lipid levels.

Direct gene transfer into donor hearts at the time of harvest

Access to the donor heart at the time of harvest provides a unique opportunity for genetic manipulation of this organ before transplantation. We sought to determine (1) if donor mouse hearts express a foreign gene administered at harvest and, (2) if so, what route of gene delivery is most effective. At harvest, 30 micrograms of promoter cytomegalovirus-luciferase deoxyribonucleic plasmid in cationic liposomes was injected directly into the myocardial apex (group I), into the right atrium (group II), or into the coronary arteries (group III). The donor hearts were then transplanted into the abdomen of recipient mice of the same strain. The transplanted hearts were removed in 4 days and luciferase expression was assayed by immunohistochemistry. In group I, luciferase activity was localized to the apex. In group II, where plasmid was delivered into the right atrium, luciferase expression was detected in the right ventricle and sparsely in the coronary perivascular area. In group III, where plasmid was injected into the coronary arteries, the transplanted hearts demonstrated luciferase expression in (1) perivascular areas surrounding coronary arteries and veins, (2) coronary capillaries, and (3) the endocardia of both ventricles. This study suggests that (1) donor mouse hearts can be genetically modified at the time of harvest and (2) intracoronary infusion of plasmid yields the most effective method of delivery. Administration of plasmid in the coronary arteries localizes the expression to the endocardium and the coronary vasculature, both sites of immunologic interactions after heart transplantation.

Involvement of the tyrosinase gene in the deposition of cardiac lipofuscin in mice. Association with aortic fatty streak development.

Lipofuscin pigment, a terminal oxidation product, accumulates within cells during the normal aging process and under certain pathological conditions. We have analyzed a genetic cross between two inbred mouse strains, BALB/cJ and a subline of C57BL/6J, which differ in lipofuscin deposition. A comparison of the segregation pattern of cardiac lipofuscin with the albino locus (c) on mouse chromosome 7 revealed complete concordance. Analysis of spontaneous mutants of the tyrosinase gene, encoded by the albino locus, confirmed that the tyrosinase gene itself controls lipofuscin formation. Genetic analysis of other strains indicated that one or more additional genes cab contribute to the inheritance of lipofuscin. We also present evidence for an association between cardiac lipofuscin deposition and aortic fatty streak development in the mouse.