Paul G. Stalboerger

Smooth Muscle Progenitor Cells in Human Blood

Background—Recent animal data suggest that vascular smooth muscle cells within the neointima of the vessel wall may originate from bone marrow, providing indirect evidence for circulating smooth muscle progenitor cells (SPCs). Evidence for circulating SPCs in human subjects does not exist, and the mechanism whereby such putative SPCs may home to sites of plaque formation is presently not understood but is likely to involve expression of specific surface adhesion molecules, such as integrins. In this study, we aimed to culture smooth muscle outgrowth cells (SOCs) from SPCs in human peripheral blood and characterize surface integrin expression on these cells. Methods and Results—Human mononuclear cells isolated from buffy coat were seeded on collagen type 1 matrix and outgrowth cells selected in endothelial growth medium (EGM-2) or EGM-2 and platelet-derived growth factor BB. Selection in platelet-derived growth factor BB–enriched medium caused rapid outgrowth and expansion of SOC to >40 population doublings in a 4-month period. These SOCs were positive for smooth muscle cell–specific &agr; actin (&agr;SMA), myosin heavy chain, and calponin on immunofluorescence and Western blotting and were also positive for CD34, Flt1, and Flk1 receptor but negative for Tie-2 receptor expression, suggesting a potential bone marrow angioblastic origin. In contrast, endothelial outgrowth cells (EOCs) grown in EGM-2 alone and the initial MNC population were negative for these smooth muscle–specific markers. Integrin &agr;5&bgr;1 expression by FACS and Western blotting was significantly increased in SOCs compared with EOCs, and this was confirmed by 8-fold greater adhesion of SOC to fibronectin (P <0.001), an effect that could be decreased using an &agr;5&bgr;1 antibody. Finally, SOC showed a significantly greater in vitro proliferative potential compared with EOCs of similar passage (P <0.001). Conclusions—This study demonstrates for the first time outgrowth of smooth muscle cells with a specific growth, adhesion, and integrin profile from putative SPC in human blood. These data have implications for our understanding of adult vascular smooth muscle cell differentiation, proliferation, and homing. (Circulation. 2002;106:1199–1204.)

Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation

Atherosclerosis is the major cause of adult mortality in the developed world, and a significant contributor to atherosclerotic plaque progression involves smooth muscle cell recruitment to the intima of the vessel wall. Controversy currently exists on the exact origin of these recruited cells. Here we use sex-mismatched bone marrow transplant subjects to show that smooth muscle cells throughout the atherosclerotic vessel wall can derive from donor bone marrow. We demonstrate extensive recruitment of these cells in diseased compared with undiseased segments and exclude cell–cell fusion events as a cause for this enrichment. These data have broad implications for our understanding of the cellular components of human atherosclerotic plaque and provide a potentially novel target for future diagnostic and therapeutic strategies.

Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation.

Abstract:  Background:  Experience with non‐antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non‐Gal endothelial cell (EC) antigens.

Cytogenetic studies of epithelial ovarian carcinoma

We performed cytogenetic studies of 36 human epithelial ovarian carcinomas using in situ culture and robotic harvest. We obtained analyzable metaphases of all 36 tumors (100%). One or more chromosomally abnormal clones were observed in 80% of tumors. Common clonal chromosome gains (each occurring in six or more cases) included +1, +2, +3, +6, +7, +9, and +12. Common clonal chromosome losses (occurring in 12 or more cases) included -X, -4, -8, -11, -13, -15, -17, and -22. Common clonal structural abnormalities (occurring in four or more cases) involved regions 1p36, 1q32, 1q42, 3p13-->p26, 3q26-->q29, 7p22, 9q34, 11p13-p15, 17q21-->q23, 19p13.3, and 19q13.3. Trisomy 12 was noted as the sole anomaly in three of five borderline and grade 1 tumors. Two grade 2 tumors contained i(1q), -14, -15 and -22. The results suggest that the pathogenesis of borderline and low-grade tumors may differ from that of higher grade tumors. Two high-grade tumors had an apparent translocation between 17q21 and 19p13.3, two chromosome regions believed to be critical to ovarian carcinogenesis.

Progenitor Cell Therapy in a Porcine Acute Myocardial Infarction Model Induces Cardiac Hypertrophy, Mediated by Paracrine Secretion of Cardiotrophic Factors Including TGFβ1

Administration of endothelial progenitor cells (EPC) is a promising therapy for post-infarction cardiac repair. However, the mechanisms that underlie apparent beneficial effects on myocardial remodeling are unclear. In a porcine model of acute myocardial infarction, we investigated the therapeutic effects of a mixed population of culture modified peripheral blood mononuclear cells (termed hereafter porcine EPC). Porcine EPC were isolated using methods identical to those previously adopted for harvest of EPC in human cell therapy studies. In addition the therapeutic effects of paracrine factors secreted by these cells was evaluated in vitro and in vivo. Intracoronary injection of autologous porcine EPC was associated with increased infarct territory mass and improved regional ventricular systolic function at 2 months compared to control. Treatment with conditioned media derived from autologous EPC was associated with similar improved effects on infarct territory mass and function. Histologic analysis of the infarct territory revealed significantly increased cardiomyocyte size in EPC and conditioned media treated groups, when compared to controls. A paracrine EPC effect was also verified in a pure myocardial preparation in which cardiomyocytes devoid of fibroblast, neuronal and vascular elements directly responded by increasing cell mass when exposed to the same conditioned media. Analysis of conditioned media revealed elevated levels of TGFbeta1 (human 267.3+/-11.8 pg/ml, porcine 57.1+/-6.1 pg/ml), a recognized mediator of hypertrophic signaling in the heart. Neutralizing antibodies to TGFbeta1 attenuated the pro-hypertrophic effect of conditioned media, and use of recombinant TGFbeta1 added to fresh media replicated the pro-hypertrophic effects of conditioned media in vitro. These data demonstrate the potential of paracrine factors secreted from endothelial progenitor cells to induce cardiomyocyte hypertrophy contributing to increased infarct territory LV mass, with favorable medium term effects on regional function following myocardial infarction.

Identification of New Carbohydrate and Membrane Protein Antigens in Cardiac Xenotransplantation

Background. &agr;1,3-Galactosyltransferase gene knockout (GTKO) pigs reduced the significance of antibody to galactose alpha 1,3-galactose (Gal) antigens but did not eliminate delayed xenograft rejection (DXR). We hypothesize that DXR of GTKO organs results from an antibody response to a limited number of non-Gal endothelial cell (EC) membrane antigens. In this study, we screened a retrovirus expression library to identify EC membrane antigens detected after cardiac xenotransplantation. Methods. Expression libraries were made from GT+:CD46 and GTKO porcine aortic ECs. Viral stocks were used to infect human embryonic kidney cells (HEK) that were selected by flow cytometry for IgG binding from sensitized cardiac heterotopic xenograft recipients. After three to seven rounds of selection, individual clones were assessed for non-Gal IgG binding. The porcine complementary DNA was recovered by polymerase chain reaction amplification, sequenced, and identified by homology comparisons. Results. A total of 199 and 317 clones were analyzed from GT+:CD46 and GTKO porcine aortic EC complementary DNA libraries, respectively. Sequence analysis identified porcine CD9, CD46, CD59, and the EC protein C receptor. We also identified porcine annexin A2 and a glycosyltransferase with homology to the human &bgr;1,4 N-acetylgalactosaminyl transferase 2 gene. Conclusion. The identified proteins include key EC functions and suggest that non-Gal antibody responses may compromise EC functions and thereby contribute to DXR. Recovery of the porcine &bgr;1,4 N-acetylgalactosaminyl transferase 2 suggests that an antibody response to a SDa-like carbohydrate may represent a new carbohydrate moiety involved in xenotransplantation. The identification of these porcine gene products may lead to further donor modification to enhance resistance to DXR and further reduce the level of xenograft antigenicity.

Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts.

Background. Transgenic expression of human complement regulatory proteins reduces the frequency of hyperacute rejection (HAR) in Gal-positive cardiac xenotransplantation. In this study, we examined the impact of human CD55 (hCD55) expression on a Gal knockout (GTKO) background using pig-to-primate heterotopic cardiac xenotransplantation. Methods. Cardiac xenotransplantation was performed with GTKO (group 1; n=6) and GTKO.hCD55 (group 2; n=5) donor pigs using similar immunosuppression. Cardiac biopsies were obtained 30 min after organ reperfusion. Rejection was characterized by histology and immunohistology. Intragraft gene expression, serum non-Gal antibody, and antibody recovered from rejected hearts were analyzed. Results. HAR of a GTKO heart was observed. Remaining grafts developed delayed xenograft rejection. Median survival was 21 and 28 days for groups 1 and 2, respectively. Vascular antibody deposition was uniformly detected 30 min after organ reperfusion and at explant. A higher frequency of vascular C5b deposition was seen in GTKO organs at explant. Serum non-Gal antibody, antibody recovered from the graft, and intragraft gene expression were similar between the groups. Conclusion. HAR of GTKO hearts without hCD55 may occur. Expression of hCD55 seemed to restrict local complement activation but did not improve graft survival. Chronic vascular antibody deposition with evidence of protracted endothelial cell activation was seen. These observations suggest that non-Gal antibody-induced chronic endothelial cell activation coupled to possible hemostatic incompatibilities may be the primary stimulus for delayed xenograft rejection of GTKO hearts. To avoid possible HAR, future clinical studies should use donors expressing human complement regulatory proteins in the GTKO background.

Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen

Xenograft rejection of pigs organs with an engineered mutation in the GGTA‐1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non‐Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig‐to‐primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N‐acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance.

Optimized Delivery System Achieves Enhanced Endomyocardial Stem Cell Retention

Background—Regenerative cell-based therapies are associated with limited myocardial retention of delivered stem cells. The objective of this study is to develop an endocardial delivery system for enhanced cell retention. Methods and Results—Stem cell retention was simulated in silico using 1- and 3-dimensional models of tissue distortion and compliance associated with delivery. Needle designs, predicted to be optimal, were accordingly engineered using nitinol, a nickel and titanium alloy displaying shape memory and superelasticity. Biocompatibility was tested with human mesenchymal stem cells. Experimental validation was performed with species-matched cells directly delivered into Langendorff-perfused porcine hearts or administered percutaneously into the endocardium of infarcted pigs. Cell retention was quantified by flow cytometry and real-time quantitative polymerase chain reaction methodology. Models, computing optimal distribution of distortion calibrated to favor tissue compliance, predicted that a 75°-curved needle featuring small-to-large graded side holes would ensure the highest cell retention profile. In isolated hearts, the nitinol curved needle catheter (C-Cath) design ensured 3-fold superior stem cell retention compared with a standard needle. In the setting of chronic infarction, percutaneous delivery of stem cells with C-Cath yielded a 37.7±7.1% versus 10.0±2.8% retention achieved with a traditional needle without effect on biocompatibility or safety. Conclusions—Modeling-guided development of a nitinol-based curved needle delivery system with incremental side holes achieved enhanced myocardial stem cell retention.

Cytogenetic studies of four hemangiopericytomas

We report cytogenetic studies on four hemangiopericytomas using in situ culture and robotic harvesting. Simple clonal chromosome abnormalities were observed in each of the three successful tumors. A cerebral tumor had a nonconstitutional 46,XX,t(7;12)(p15;q15) karyotype. A recurrent cerebral tumor had two cell lines: one with a 46,XY,t(2;12)(p13;q24.1) karyotype and one with a 46,XY,t(5;7)(q13;p15) karyotype. Each of the metaphases from a lung tumor had a 46,Y,inv(X)(p22.3q13) karyotype. We conclude that simple nonconstitutional balanced chromosome anomalies of 7p15 and 12q are common in cerebral hemangiopericytomas. It is possible that balanced anomalies of these chromosome regions are related to the pathogenesis of this tumor type.