Soniya Nityanand

Mesenchymal stem cells (MSC) have emerged as a potential stem cell type for cardiac regeneration after myocardial infarction (MI). Recently, we isolated and characterized mesenchymal stem cells derived from rat fetal heart (fC-MSC), which exhibited potential to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. In the present study, we investigated the therapeutic efficacy of intravenously injected fC-MSC in a rat model of MI using multi-pinhole gated SPECT-CT system. fC-MSC were isolated from the hearts of Sprague Dawley (SD) rat fetuses at gestation day 16 and expanded ex vivo. One week after induction of MI, 2×10⁶ fC-MSC labeled with PKH26 dye (n = 6) or saline alone (n = 6) were injected through the tail vein of the rats. Initial in vivo tracking of 99mTc-labeled fC-MSC revealed a focal uptake of cells in the anterior mid-ventricular region of the heart. At 4 weeks of fC-MSC administration, the cells labeled with PKH26 were located in abundance in infarct/peri-infarct region and the fC-MSC treated hearts showed a significant increase in left ventricular ejection fraction and a significant decrease in the end diastolic volume, end systolic volume and left ventricular myo-mass in comparison to the saline treated group. In addition, fC-MSC treated hearts had a significantly better myocardial perfusion and attenuation in the infarct size, in comparison to the saline treated hearts. The engrafted PKH26-fC-MSC expressed cardiac troponin T, endothelial CD31 and smooth muscle sm-MHC, suggesting their differentiation into all major cells of cardiovascular lineage. The fC-MSC treated hearts demonstrated an up-regulation of cardio-protective growth factors, anti-fibrotic and anti-apoptotic molecules, highlighting that the observed left ventricular functional recovery may be due to secretion of paracrine factors by fC-MSC. Taken together, our results suggest that fC-MSC therapy may be a new therapeutic strategy for MI and multi-pinhole gated SPECT-CT system may be a useful tool to evaluate cardiac perfusion, function and cell tracking after stem cell therapy in acute myocardial injury setting.

AIM: To study the expression of embryonal markers by fetal cardiac mesenchymal stem cells (fC-MSC) and their differentiation into cells of all the germ layers. METHODS: Ten independent cultures of rat fC-MSC were set up from cells derived from individual or pooled fetal hearts and studies given below were carried out at passages 3, 6, 15 and 21. The phenotypic markers CD29, CD31, CD34, CD45, CD73, CD90, CD105, CD166 and HLA-DR were analyzed by flow cytometry. The expression of embryonal markers Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA 1-81 were studied by immunocytochemistry. The fC-MSC treated with specific induction medium were evaluated for their differentiation into (1) adipocytes and osteocytes (mesodermal cells) by Oil Red O and Alizarin Red staining, respectively, as well as by expression of lipoprotein lipase, PPARγ2 genes in adipocytes and osteopontin and RUNX2 genes in osteocytes by reverse-transcription polymerase chain reaction (RT-PCR); (2) neuronal (ectodermal) cells by expression of neuronal Filament-160 and Glial Fibrillar Acidic Protein by RT-PCR and immunocytochemistry; and (3) hepatocytic (endodermal) cells by expression of albumin by RT-PCR and immunocytochemistry, glycogen deposits by Periodic Acid Schiff staining and excretion of urea into the culture supernatant. RESULTS: The fC-MSC expressed CD29, CD73, CD90, CD105, CD166 but lacked expression of CD31, CD34, CD45 and HLA-DR. They expressed embryonal markers, viz. Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-81 but not TRA-1-60. On treatment with specific induction media, they differentiated into adipocytes and osteocytes, neuronal cells and hepatocytic cells. CONCLUSION: Our results together suggest that fC-MSC are primitive stem cell types with a high degree of plasticity and, in addition to their suitability for cardiovascular regenerative therapy, they may have a wide spectrum of therapeutic applications in regenerative medicine.

To investigate soluble (s) E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1), and their relationship to disease activity in Takayasu's arteritis (TA). Levels of adhesion molecules were measured by enzyme immunoassay in the sera of 35 patients with TA, 17 healthy controls, and 15 patients with 12 months followup. Compared to controls, patients had elevated levels of sE-selectin (54.5 +/- 35.0 vs 36.4 +/- 13.0 ng/ml, p < 0.05), sVCAM-1 (280.9 +/- 267.6 vs 141.2 +/- 76.1 ng/ml, p < 0.05), and sICAM-1 (261.3 +/- 168.1 vs 198.3 +/- 74.3 ng/ml, p < 0.05). Compared to controls, patients with inactive TA also had elevated levels of sE-selectin (67.4 +/- 45.9 vs 36.4 +/- 13.0 ng/ml, p < 0.02), sVCAM-1 (327.6 +/- 327.8 vs 141.2 +/- 76.1 ng/ml, p < 0.02), and sICAM-1 (321.9 +/- 179.5 vs 198.3 +/- 74.3 ng/ml, p < 0.02). There was no difference between active TA and controls. sE-selectin had a trend towards increased levels in inactive versus active TA (67.4 +/- 45.9 vs 44.9 +/- 20.3 ng/ml p = 0.059), but there was no difference in sVCAM-1 and sICAM-1 levels between the groups. No adhesion molecule levels showed a change among followup patients. Patients with inactive TA have elevated levels of sE-selectin, sVCAM-1, and sICAM-1 that might indicate persistent vasculopathy in clinically inactive disease.

Objective: Increased numbers of circulating γ/δ T cells with a restricted T cell receptor repertoire, as well as colocalization of the expression of heat‐shock protein Hsp60/65 and γ/δ T cells in the arterial lesions of patients with Takayasu arteritis (TA), indicate that γ/δ T cells may react to Hsp60 and cause damage to the arterial endothelium. In this study we investigated the proliferative responses of γ/δ T cells to human Hsp60 and their cytotoxicity to human aortic endothelial cells (ECs) in patients with TA.Methods: Blood samples were obtained from 12 patients with TA, 8 patients with systemic lupus erythematosus (SLE) (as disease controls), and 10 healthy control subjects. Proliferative responses of circulating γ/δ T cells to human Hsp60 were detected by flow cytometry–based bromodeoxyuridine incorporation assay. Cytotoxicity of the γ/δ T cells to human aortic ECs was analyzed by colorimetric lactate dehydrogenase release assay.Results: The γ/δ T cells of 11 of 12 patients with TA exhibited reactivity to Hsp60, whereas none of the γ/δ T cells from patients with SLE or healthy controls showed reactivity (both P < 0.001). The mean ± SD proliferative response of γ/δ T cells in patients with TA was 21.4 ± 11.3%, compared with 4.2 ± 1.2% in patients with SLE and 4.01 ± 1.82% in healthy controls (both P < 0.001). In addition, compared with the control groups, the γ/δ T cells of patients with TA had increased spontaneous cytotoxicity to aortic ECs (22.1 ± 15.0% versus 9.6 ± 2.13% in SLE patients and 8.1 ± 4.7% in healthy controls; both P < 0.005), which was further enhanced following stimulation of γ/δ T cells with Hsp60. The cytotoxicity of the γ/δ T cells was significantly inhibited by treatment of these cells with concanamycin A and anti–Fas ligand–blocking antibodies.Conclusion: The results show that γ/δ T cells in patients with TA are reactive to Hsp60 and exhibit cytotoxicity to aortic ECs, suggesting a key role of Hsp60 and γ/δ T cells in the pathogenesis of TA.

Objective: Anti–endothelial cell antibodies are considered to have an important role in the pathogenesis of Takayasu arteritis (TA). Previously, these antibodies were detected using human umbilical vein endothelial cells, which do not completely represent the antigenicity/functions of aortic endothelial cells, the specific targets in TA. To delineate the precise role of antigenic targets, we investigated such targets as well as the pathogenic mechanism of antibodies directed against aortic endothelial cells (AAECAs) in TA.Methods: AAECAs were detected using a cellular enzyme‐linked immunosorbent assay (ELISA), and their antigenic targets were detected by immunoblotting. AAECA‐mediated induction of endothelial adhesion molecule expression and cytokine production was studied by ELISA, and apoptosis was studied using the TUNEL method.Results: AAECAs were detected in 86% of patients with TA and in 9% of controls. Sera obtained from AAECA‐positive patients with TA recognized a total of 9 antigens ranging in size from 18 kd to 200 kd, of which the 60–65‐kd triplet was recognized most often. The aortic endothelial cell reactivity of Hsp60‐absorbed sera was reduced by ∼50% as compared with that of unabsorbed sera (mean ± SD 0.488 ± 0.08 versus 0.838 ± 0.116). Sera from AAECA‐positive patients with TA, compared with sera from AAECA‐negative patients with TA and that from controls, induced increased expression of E‐selectin (mean ± SD 0.833 ± 0.063 versus 0.217 ± 0.081 and 0.221 ± 0.101 optical density [OD] units, respectively) and vascular cell adhesion molecule 1 (0.620 ± 0.144 versus 0.165 ± 0.005 and 0.177 ± 0.055 OD units, respectively) and increased production of interleukin‐4 (IL‐4) (6.8 ± 2.4 versus 1.2 ± 1.6 and 1.3 ± 2.5 pg/ml, respectively), IL‐6 (24.3 ± 2.4 versus 4.5 ± 6.7 and 5.9 ± 5.1 pg/ml, respectively), and IL‐8 (36.8 ± 10.3 versus 10.1 ± 6.7 and 7.8 ± 2.1 pg/ml, respectively). Sera from AAECA‐positive patients with TA induced 29 ± 6% (median ± SEM) apoptosis of aortic endothelial cells.Conclusion: Our data show that the AAECAs that are present in patients with TA are directed mainly against 60–65‐kd antigen(s) and may cause vascular dysfunction by inducing expression of endothelial adhesion molecules, cytokine production, and apoptosis.

Interleukin-8 (IL-8), a CXC chemokine, is also a potent inhibitor of myelopoiesis, the hematopoietic process that is severely impaired in aplastic anemia (AA). To elucidate its role in the disease, we have investigated levels of IL-8 by quantitative enzyme-linked immunosorbent assay in bone marrow and peripheral blood plasma of 27 AA patients and in the marrow of 16 controls and blood of 20 controls. Significantly increased levels of IL-8 were observed in the marrow and blood of patients as compared to controls (470.4 ± 549.6 vs. 37.5 ± 30.3; P < 0.0001) and (247.3 ± 286.3 vs. 7.9 ± 5.5; P < 0.0001), respectively. Among the patients, the IL-8 levels were higher in patients with severe AA than those with nonsevere AA in the marrow (568.8 ± 586.9 vs. 126.3 ± 102.5; P < 0.005) as well as in the blood (296.6 ± 305.5 vs. 75.0 ± 84.4; P < 0.008) plasma. The marrow and blood of 74% (20/27) of the patients had increased levels of IL-8 compared to 12% (2/16; P < 0.001) and 10% (2/20, P < 0.001) of the controls, respectively. These results suggest that IL-8 may have an important role in the pathogenesis of AA. Am. J. Hematol. 79:240–242, 2005.

Immune-mediated stem cell damage has been postulated to be responsible for disease initiation and progression in aplastic anemia (AA). It is hypothesized that T lymphocytes play a major role in destroying the bone marrow (BM) stem cells of AA patients by infiltrating the BM and secreting excessive levels of anti-hematopoietic cytokines, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). We undertook this study to assess the pathogenic significance of anti-hematopoietic cytokines such as IFN-γ and TNF-α in BM T cells and plasma of AA patients. Significantly elevated levels of IFN-γ and TNF-α were found in the BM plasma of AA patients compared to controls (p=0.05 and 0.006, respectively). Intracellular IFN-γ and not TNF-α in BM CD3⁺ T cells of AA patients was significantly higher compared to controls (p=0.04 and p=0.2, respectively). A follow-up analysis of expression of these cytokines in BM T cells and their levels in BM plasma in five AA patients before and 180 days (6 months) after antithymocyte globulin (ATG) and cyclosporin A (CsA) therapy showed a decline 180 days after therapy compared to pre-therapy. We thus conclude that increased production of both IFN-γ and TNF-α in the BM may contribute to disease pathogenesis in AA and ATG therapy may induce hematological remission by suppressing the elevated levels of IFN-γ and TNF-α in AA BM.

Expression of heat shock protein (HSP)‐65 as well as infiltration of T‐cells in arterial lesions and raised levels of circulating antibodies against mycobacterial HSP65 (mHSP65) led us to the concept that mHSP65 or its human homologue (hHSP60) might be involved in the etiopathogenesis of Takayasu's arteritis (TA). Therefore, we investigated mHSP65 and hHSP60 reactive peripheral blood T‐cell subsets by BrdU incorporation assay and flow cytometry as well as investigating the different isotypes of anti‐mHSP65 and hHSP60 antibodies by ELISA. Eighty‐four percent (22/26) of the TA patients were observed to show T‐cell proliferation to mHSP65 and hHSP60 whereas only 16% (3/18) healthy controls showed such proliferation (P < 0·001). Both HSPs induced proliferation of exclusively CD4+ T‐cells and not CD8+ T‐cells. We also observed a significantly higher prevalence of only the IgG isotype reactive to mHSP65 and hHSP60 in TA as compared to HC (mHSP65: 92% TA versus 11% HC, P < 0·0001 and hHSP60: 84% versus 22%, P < 0·001). Our data show a significant correlation between mHSP65 and hHSP60 reactive T‐cells (CD3+: r = 0·901; CD4+: r = 0·968) as well as anti‐mHSP65 and anti‐hHSP60 IgG antibodies (r = 0·814) suggesting an infection induced autoimmunity in TA, possibly induced by molecular mimicry between mHSP65 and hHSP60 or other tissue specific antigens.

SUMMARY: We have investigated constitutive and phytohaemagglutinin (PHA) + phorbol 12-myristate 13-acetate (PMA)-induced gene expression of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, IL-3, IL-4, IL-10, IL-12 and granulocyte macrophage colony-stimulating factor (GM-CSF) in peripheral blood mononuclear cells (PBMCs) of 10 patients with Takayasu's arteritis (TA) and 10 healthy controls by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). The constitutive mRNA expression of TNF-α (69·0 ± 4·0%versus 27·5 ± 18·0%; P = 0·001) and IL-4 (60·0 ± 10·0%versus 0%; P = 0·001) was significantly higher in patients than controls; that of IL-3 was comparable in both groups (38·0 ± 6·0%versus 32·0 ± 5·0%; P = 0·651) while no constitutive mRNA expression was observed for the other cytokines studied. The stimulated PBMCs of patients, as compared with the controls, had higher mRNA gene expression of TNF-α (127·0 ± 16·0%versus 54·0 ± 6·0%; P = 0·001), IFN-γ (93·0 ± 13·0%versus 57·0 ± 5·0%; P = 0·032), IL-2 (109·0 ± 13·0%versus 68·0 ± 6·0%; P = 0·015), IL-3 (60·0 ± 8·0%versus 21·2 ± 3·0%; P = 0·045) and IL-4 (68·0 ± 7·0%versus 27·0 ± 7·2%; P = 0·01) The mRNA expression of IL-10 was lower in patients than controls (35·0 ± 8·0%versus 75·0 ± 12·0%; P = 0·022). The GM-CSF mRNA was similar (102·0 ± 6·0%versus 89·0 ± 5·0%; P = 0·475) in both groups. Stimulation of cells with PHA + PMA showed no IL-12 expression but stimulation with lipopolysaccharide induced higher IL-12 mRNA in patients than controls (83·0 ± 14·0%versus 33·0 ± 4·0%; P = 0·005). Our data suggest that an inflammatory cytokine signature exists in TA with a key role for TNF-α, IL-4, IL-10 and IL-12 in different pathological processes of the disease.

Annexin V has an important role in the regulation of apoptosis and antibodies directed against it have been shown to lead to apoptosis of vascular endothelial cells. To evaluate the role of anti‐annexin V antibodies (AA5A) in Takayasu's arteritis (TA), we investigated these antibodies in the sera of 66 TA patients, 50 healthy controls and in the follow‐up sera of 12 active TA patients by enzyme‐linked immunosorbent assay. The AA5A‐positive patients were analysed further for the presence of anti‐endothelial cell antibodies (AECA) and anticardiolipin antibodies (ACLA) to determine the relationship of AA5A with these autoantibodies. AA5A were observed in 36% (24/66) of the patients versus 6% (3/50) of the controls (P < 0·001) and in 53% (19/36) of patients with active TA versus 17% (5/30) of those with inactive disease (P < 0·01). Levels of AA5A were also observed to be significantly higher in patients with TA compared to controls (0·557 ± 0·362 versus 0·259 ± 0·069; P < 0·0001) and in patients with active disease compared to those with inactive disease (0·700 ± 0·403 versus 0·385 ± 0·205; P < 0·0001). In the follow‐up study, 6/12 patients who became inactive during follow‐up also showed normalization of AA5A levels. AECA and ACLA were detected in 54% (13/24) and 12% (3/24) of the AA5A‐positive patients, respectively. Our results show that a significant proportion of TA patients have AA5A, which exhibit an association with AECA and because they have a correlation with disease activity thus appear to be involved in the disease pathogenesis.