α1,3-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.
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.
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 β1,4 N-acetylgalactosaminyl transferase 2 gene.
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 β1,4 N-acetylgalactosaminyl transferase 2 suggests that an antibody response to a SD-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.
Current options for in vivo regeneration of dermal tissue remain limited. The purpose of this study was to engineer a unique scaffold capable of recruiting dermal stem cells from adjacent tissue, ...thus circumventing the need to seed the scaffolds with stem cells before implantation, leading to skin regeneration.
A hydrogel scaffold was created through combination of type I collagen along with fractionated platelet-rich plasma. This was compared to a control hydrogel consisting of type I collagen and fetal bovine serum. Hydrogels were cultured with fresh human skin tissue and incubated with supplemental media. Gels were digested weekly for cellular content as examined by flow cytometry at the 4- and 8-week time points. The fractionated platelet-rich plasma and collagen gels were then implanted onto full-thickness skin defects on the backs of rats and compared to wounds healing by secondary intention. Wound area was evaluated for epithelialization and neovascularization.
Platelet-rich plasma fractionation increased platelet-derived growth factors. In contrast to collagen scaffolds, fractionated platelet-rich plasma-supplemented scaffolds recruited more dermal-derived stem cells from fresh skin tissue compared with collagen hydrogels at the 4- and 8-week time points. Furthermore, fractionated platelet-rich plasma-supplemented hydrogels accelerated wound healing, angiogenesis, and hair and sweat gland formation, ultimately regenerating a dermis-like tissue.
Generation of hydrogels with fractionated platelet-rich plasma was able to improve cellular recruitment and growth and differentiation of dermal-derived stem cells, leading to hair growth and sweat gland formation. This provides a novel approach to regenerate skin for treating large defects.
Background
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.
Methods
The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK‐B4T) was produced. Expression of porcine B4GALNT2 in HEK‐B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK‐B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK‐B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non‐Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining.
Results
The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes, respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK‐B4T) results in increased binding of antibody to the B4GALNT2 enzyme, and increased reactivity with anti‐Sda and DBA. HEK‐B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non‐Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK‐B4T cells.
Conclusion
The functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK‐B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non‐Gal glycan which contributes in part to the non‐Gal immune response detected after pig‐to‐baboon cardiac xenotransplantation.
Smooth muscle progenitor cells in human blood Simper, David; Stalboerger, Paul G; Panetta, Carmelo J ...
Circulation (New York, N.Y.),
09/2002, Letnik:
106, Številka:
10
Journal Article
Recenzirano
Odprti dostop
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.
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 alpha actin (alphaSMA), 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 alpha5beta1 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 alpha5beta1 antibody. Finally, SOC showed a significantly greater in vitro proliferative potential compared with EOCs of similar passage (P<0.001).
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.
The application of brief high voltage electrical pulses to tissue can lead to an irreversible or reversible electroporation effect in a cell-specific manner. In the management of ventricular ...arrhythmias, the ability to target different tissue types, specifically cardiac conduction tissue (His-Purkinje System) vs. cardiac myocardium would be advantageous. We hypothesize that pulsed electric fields (PEFs) can be applied safely to the beating heart through a catheter-based approach, and we tested whether the superficial Purkinje cells can be targeted with PEFs without injury to underlying myocardial tissue.
In an acute (n = 5) and chronic canine model (n = 6), detailed electroanatomical mapping of the left ventricle identified electrical signals from myocardial and overlying Purkinje tissue. Electroporation was effected via percutaneous catheter-based Intracardiac bipolar current delivery in the anesthetized animal. Repeat Intracardiac electrical mapping of the heart was performed at acute and chronic time points; followed by histological analysis to assess effects.
PEF demonstrated an acute dose-dependent functional effect on Purkinje, with titration of pulse duration and/or voltage associated with successful acute Purkinje damage. Electrical conduction in the insulated bundle of His (n = 2) and anterior fascicle bundle (n = 2), was not affected. At 30 days repeat cardiac mapping demonstrated resilient, normal electrical conduction throughout the targeted area with no significant change in myocardial amplitude (pre 5.9 ± 1.8 mV, 30 days 5.4 ± 1.2 mV, p = 0.92). Histopathological analysis confirmed acute Purkinje fiber targeting, with chronic studies showing normal Purkinje fibers, with minimal subendocardial myocardial fibrosis.
PEF provides a novel, safe method for non-thermal acute modulation of the Purkinje fibers without significant injury to the underlying myocardium. Future optimization of this energy delivery is required to optimize conditions so that selective electroporation can be utilized in humans the treatment of cardiac disease.
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.
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.
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.
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.
Regenerative Medicine
Extracellular vesicles from healthy human platelets globally control inflammation in the hearts of mice with viral myocarditis by regulating the innate immune response with ...their heterogenous functional contents. This immunomodulation in part comes from anti‐inflammatory microRNAs and prevents cardiac dysfunction during peak acute myocarditis. The therapeutic tested in article number 2303317 by DeLisa Fairweather and co‐workers offers a novel and translatable option for inflammatory cardiovascular diseases.
Background
Bone marrow-derived mesenchymal stem cells (bmMSCs) have been used as a cellular therapeutic option for treatment of osteonecrosis of the femoral head. However, use of bmMSCs as a ...treatment adjuvant for orthopaedic disorders in general has achieved limited success. Adipose-derived MSCs (aMSCs) may be a more-efficient regenerative cell source given their greater quantity and protection from physiologic stress.
Questions
/
purposes
We asked the following questions in a paired analysis of MSCs from patients with osteonecrosis: (1) Is there a difference in proliferation potential between aMSCs and bmMSCs? (2) Is there a difference in osteogenic differentiation potential between aMSCs and bmMSCs? (3) Are genetic pathways differentially expressed between aMSCs and bmMSCs that may govern functional phenotypic discrepancies?
Methods
Periarticular samples of adipose tissue and bone marrow from the femoral canal were obtained from 15 patients undergoing hip replacement for late-stage (Steinberg Stages III-VI) osteonecrosis. MSCs were isolated from both tissue sources and taken through a standardized 20-day cell division protocol to establish cumulative cell count. They also were grown in osteogenic differentiation media for 14 days with subsequent measurement of alkaline phosphatase in units of optical density. RNA was isolated from aMSCs and bmMSCs in five patients to assess differentially expressed genetic pathways using the Affymetrix GeneChip
®
Human Transcriptome Array 2.0 platform.
Results
Proliferation capacity was increased by fourfold in aMSCs compared with bmMSCs after 20 days in culture. The mean difference in cumulative cell count was 3.99 × 10
8
cells (SD = 1.67 × 10
8
cells; 95% CI, 3.07 × 10
8
–4.92 × 10
8
cells; p < 0.001). Bone differentiation efficiency as measured by optical density was increased by 2.25-fold in aMSCs compared with bmMSCs. The mean difference in optical density was 1.27 (SD = 0.34; 95% CI, 1.08–1.46; p < 0.001). RNA transcriptome analysis showed 284 genes that met statistical (p < 0.05) and biological (fold change > 1.5) significance cutoffs for differential expression between cell populations. Subsequent network topology of differentially expressed genes showed alterations in pathways critical for musculoskeletal tissue development in addition to many nonspecific findings.
Conclusions
aMSCs outperform bmMSCs in growth rate and bone differentiation potential in the setting of osteonecrosis, suggesting they may provide a more-potent regenerative therapeutic strategy in this population. Differential expression of genes and cellular pathways highlighted in this study may provide therapeutic targets for cellular optimization or acellular treatment strategies.
Clinical Relevance
aMSCs may provide a more robust cellular therapeutic than bmMSCs for treatment of osteonecrosis. Ideally, a well-designed prospective study will be able to evaluate the efficacy of these cellular therapies side-by-side in patients with bilateral early stage disease.
Patients with viral myocarditis are at risk of sudden death and may progress to dilated cardiomyopathy (DCM). Currently, no disease‐specific therapies exist to treat viral myocarditis. Here it is ...examined whether reconstituted, lyophilized extracellular vesicles (EVs) from platelets from healthy men and women reduce acute or chronic myocarditis in male mice. Human‐platelet‐derived EVs (PEV) do not cause toxicity, damage, or inflammation in naïve mice. PEV administered during the innate immune response significantly reduces myocarditis with fewer epidermal growth factor (EGF)‐like module‐containing mucin‐like hormone receptor‐like 1 (F4/80) macrophages, T cells (cluster of differentiation molecules 4 and 8, CD4 and CD8), and mast cells, and improved cardiac function. Innate immune mediators known to increase myocarditis are decreased by innate PEV treatment including Toll‐like receptor (TLR)4 and complement. PEV also significantly reduces perivascular fibrosis and remodeling including interleukin 1 beta (IL‐1β), transforming growth factor‐beta 1, matrix metalloproteinase, collagen genes, and mast cell degranulation. PEV given at days 7–9 after infection reduces myocarditis and improves cardiac function. MicroRNA (miR) sequencing reveals that PEV contains miRs that decrease viral replication, TLR4 signaling, and T‐cell activation. These data show that EVs from the platelets of healthy individuals can significantly reduce myocarditis and improve cardiac function.
Extracellular vesicles from platelets of healthy adults inhibit heart inflammation when administered early or during myocarditis in a mouse model of myocarditis. This treatment globally inhibits complement, inflammasome, and T‐cell responses in the heart, as well as improving cardiac function during peak myocarditis. This novel, stable product provides immunotherapy without immunosuppression, a current need in the cardiovascular disease field.
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.
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