HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.

High-mobility group box 1 (HMGB-1) is a strong chemo-attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB-1-mediated adhesion and rolling of c-kit(+) cells and assess whether toll-like receptor-2 (TLR-2) and toll-like receptor-4 (TLR-4) of endothelial cells or c-kit(+) cells are implicated in the activation of downstream migration signals to peripheral c-kit(+) cells. Effects of HMGB-1 on the c-kit(+) cells/endothelial interaction were evaluated by a cremaster muscle model in wild-type (WT), TLR-2 (-/-) and Tlr4 (LPS-del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real-time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB-1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in 'WT' versus 9.9 ± 3.2% in 'control', P < 0.05). The number of firmly adherent c-kit(+) cells was more than 13-fold higher than that of the control group (14.6 ± 5.1 cells/mm(2) in 'WT' versus 1.1 ± 1.0 cells/mm(2) in 'control', P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR-2 (-/-) and Tlr4 (LPS-del) mice compared to WT mice (1.5 ± 1.4 cells/mm(2) in 'TLR-2 (-/-)' and 2.4 ± 1.4 cells/mm(2) in 'Tlr4 (LPS-del)' versus 14.6 ± 5.1 cells/mm(2) in 'WT', P < 0.05). TLR-2 (-/-) and Tlr4 (LPS-del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB-1 mediates c-kit(+) cell recruitment via endothelial TLR-2 and TLR-4.

Erythropoietin attenuates the sequels of ischaemic spinal cord injury with enhanced recruitment of CD34+ cells in mice.

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34(+) cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34(+) cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34(+) cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.

Mesenchymal stem cells restore lung function by recruiting resident and nonresident proteins.

Because human lungs are unlikely to repair or regenerate beyond the cellular level, cell therapy has not previously been considered for chronic irreversible obstructive lung diseases. To explore whether cell therapy can restore lung function, we administered allogenic intratracheal mesenchymal stem cells (MSCs) in the trachea of rats with chronic thromboembolic pulmonary hypertension (CTEPH), a disease characterized by single or recurrent pulmonary thromboembolic obliteration and progressive pulmonary vascular remodeling. MSCs were retrieved only in high pressure-exposed lungs recruited via a homing stromal derived factor-1α/CXCR4 pathway. After MSC administration, a marked and long-lasting improvement of all clinical parameters and a significant change of the proteome level were detected. Beside a variation of liver proteome, such as caspase-3, NF-κB, collagen1A1, and α-SMA, we also identified more than 300 resident and nonresident lung proteins [e.g., myosin light chain 3 (P16409) or mitochondrial ATP synthase subunit alpha (P15999)]. These results suggest that cell therapy restores lung function and the therapeutic effects of MSCs may be related to protein-based tissue reconstituting effects.

Cell origin of human mesenchymal stem cells determines a different healing performance in cardiac regeneration.

The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from umbilical cord blood (CB), adipose tissue (AT) or bone marrow (BM) for the treatment of myocardial infarction (MI) remains unexplored. This study was to assess the regenerative potential of hMSC from different origins and to evaluate the role of CD105 in cardiac regeneration. Male SCID mice underwent LAD-ligation and received the respective cell type (400.000/per animal) intramyocardially. Six weeks post infarction, cardiac catheterization showed significant preservation of left ventricular functions in BM and CD105(+)-CB treated groups compared to CB and nontreated MI group (MI-C). Cell survival analyzed by quantitative real time PCR for human GAPDH and capillary density measured by immunostaining showed consistent results. Furthermore, cardiac remodeling can be significantly attenuated by BM-hMSC compared to MI-C. Under hypoxic conditions in vitro, remarkably increased extracellular acidification and apoptosis has been detected from CB-hMSC compared to BM and CD105 purified CB-derived hMSC. Our findings suggests that hMSC originating from different sources showed a different healing performance in cardiac regeneration and CD105(+) hMSC exhibited a favorable survival pattern in infarcted hearts, which translates into a more robust preservation of cardiac function.

Intracardiac injection of matrigel induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model.

Matrigel promotes angiogenesis in the myocardium from ischemic injury and prevents remodelling of the left ventricle. We assessed the therapeutic efficacy of intracardiac matrigel injection and matrigel-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, matrigel (250 µl) or phosphate-buffered solution (PBS) was delivered by intracardiac injection. Compared to the MI control group (MI-PBS), matrigel significantly improved left ventricular function (n= 11, P < 0.05) assessed by pressure-volume loops after 4 weeks. There is no significant difference in infarct size between MI-matrigel (MI-M; 21.48 ± 1.49%, n = 10) and MI-PBS hearts (20.98 ± 1.25%, n = 10). The infarct wall thickness of left ventricle is significantly higher (P < 0.01) in MI-M (0.72 ± 0.02 mm, n = 10) compared with MI-PBS (0.62 ± 0.02 mm, n = 10). MI-M hearts exhibited higher capillary density (border 130.8 ± 4.7 versus 115.4 ± 6.0, P < 0.05; vessels per high-power field [HPF; 400×], n = 6) than MI-PBS hearts. c-Kit(+) stem cells (38.3 ± 5.3 versus 25.7 ± 1.5 c-Kit(+) cells per HPF [630×], n = 5, P < 0.05) and CD34(+) cells (13.0 ± 1.51 versus 5.6 ± 0.68 CD34(+) cells per HPF [630×], n = 5, P < 0.01) were significantly more numerous in MI-M than in MI-PBS in the infarcted hearts (n = 5, P < 0.05). Intracardiac matrigel injection restores myocardial functions following MI, which may attribute to the improved recruitment of CD34(+) and c-Kit(+) stem cells.

Localized SDF-1alpha gene release mediated by collagen substrate induces CD117 stem cells homing.

Stromal cell-derived factor-1alpha (SDF-1alpha) mediated mobilization and homing of stem cells showed promising potential in stem cell based tissue engineering and regenerative medicine. However local and sustained release of SDF-1alpha is indispensable for stem cell mediated regenerative process due to its short half-life under inflammatory conditions. In this study, a gene activated collagen substrate (GAC) was formed via assembly of plasmid encoding SDF-1alpha into a collagen substrate to create a microenvironment favoring stem cell homing. Local release of SDF-1alpha from the transfected cells on GAC and its effect on CD117(+) stem cell homing were investigated. Non-viral poly-ethyleneimine (25kDa PEI)/DNA complexes were mixed with rat tail collagen solution to form the GAC. Optimization of GAC was carried out based on collagen effects on the PEI/DNA complexes, viability and luciferase expression of COS7 cells on GAC. CD117(+) stem cells homing in response to SDF-1alpha local expression from transfected cells on GAC were investigated in a flow chamber in vitro and in a mouse hind limb model in vivo. The gene expression, migration of CD117(+) stem cells and the induced inflammation were investigated with immunostaining, reverse transcription polymerase chain reaction (RT-PCR) and H&E staining. The optimized parameters for GAC were DNA dosage 10 microg/cm(2), molar ratio of PEI nitrogen in primary amine to DNA phosphate (N/P ratio) 4 and mass ratio of collagen to DNA (C/D ratio) 1.0. It kept cell viability above 75% and transfection efficiency around 5.8 x 10(5) RLU/mg protein. GAC allowed the sustained gene release up to 60 days. GAC mediated SDF-1alpha gene release induced migration and homing of CD117(+) stem cells in vitro and in vivo significantly, and the inflammation of GAC reduced significantly two weeks after transplantation. GAC is a promising stem cell based therapeutic strategy for regenerative medicine.

A transformed cell population derived from cultured mesenchymal stem cells has no functional effect after transplantation into the injured heart.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells characterized by their self-renewal and differentiation potential. Accumulating clinical and preclinical evidence indicate MSCs are a promising cell source for regenerative medical therapies. However, undesirable immortalization, spontaneous transformation, and tumorigenic potential from long-term cultured MSCs have been reported in human and mouse. We report rat MSCs isolated from young donors could undergo transformation in early passage culture. We aimed to characterize the transformed population and determine their therapeutic effects after intracardiac transplantation in the infarcted myocardium. MSCs were isolated from bone marrow of Lewis rats according to standard protocols and cultured under standard conditions. Phenotype of growing cells was assessed by flow cytometry. Following acute myocardial infarction in rats, cells were delivered by intracardiac injection. Cardiac functions were assessed by pressure-volume loops. Infarction size and pathologic effects were evaluated after 6 weeks. The abnormal colonies were detected in culture as early at passage 3. They were noted to appear as distinctly different morphology from typical MSCs, which changed from a normal elongated spindle shape to a compact abnormal morphology. They exhibited rapid cell proliferation. Some subclones lost contact inhibition of cell division and formed multilayer aggregates. Chromosomal instability was detected. They were devoid of surface markers CD29, CD44, CD90, and CD117. Furthermore, there was no significant improvement on infarction size and cardiac function 6 weeks after cell transplantation. Our study highlights the need for establishment of biosafety criteria in regulating culture- expanded MSCs to achieve the full clinical therapeutic benefits.

Retraction: Mesenchymal Stem Cells: Are They Suitable for Systemic Injection? An In Vitro Study.

This article is being retracted because another article was published using the same data, but under a different title, in Microvascular Research.

Autologous umbilical cord blood mononuclear cell transplantation preserves right ventricular function in a novel model of chronic right ventricular volume overload.

We aimed to evaluate the feasibility and efficacy of autologous umbilical cord blood mononuclear cell (UCMNC) transplantation on right ventricular (RV) function in a novel model of chronic RV volume overload. Four-month-old sheep (n = 20) were randomized into cell (n = 10) and control groups (n = 10). After assessment of baseline RV function by the conductance catheter method, a transannular patch (TAP) was sutured to the right ventricular outflow tract (RVOT). Following infundibulotomy the ring of the pulmonary valve was transected without cardiopulmonary bypass. UCMNC implantation (8.22 +/- 6.28 x 10(7)) in the cell group and medium injection in the control group were performed into the RV myocardium around the TAP. UCMNCs were cultured for 2 weeks after fluorescence-activated cell sorting (FACS) analysis for CD34 antigen. Transthoracic echocardiography (TTE) and computed tomography were performed after 6 weeks and 3 months, respectively. RV function was assessed 3 months postoperatively before the hearts were excised for immunohistological examinations. FACS analysis revealed 1.2 +/- 0.22% CD34(+) cells within the isolated UCMNCs from which AcLDL(+) endothelial cells were cultured in vitro. All animals survived surgery. TTE revealed grade II-III pulmonary regurgitation in both groups. Pressure-volume loops under dobutamine stress showed significantly improved RV diastolic function in the cell group (dP/dt(min): p = 0.043; E(ed): p = 0.009). CD31 staining indicated a significantly enhanced number of microvessels in the region of UCMNC implantation in the cell group (p < 0.001). No adverse tissue changes were observed. TAP augmentation and pulmonary annulus distortion without cardiopulmonary bypass constitutes a valid large animal model mimicking the surgical repair of tetralogy of Fallot. Our results indicate that the chronically volume-overloaded RV profits from autologous UCMNC implantation by enhanced diastolic properties with a probable underlying mechanism of increased angiogenesis.

Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model.

Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function (n =11-14, P < 0.05) and decreased right ventricular wall stress (n = 8, P < 0.05) assessed by pressure-volume loops after 6 weeks. MI-EPO hearts exhibited smaller infarction size (20.1 +/- 1.1% versus 27.8 +/- 1.2%; n = 6-8, P < 0.001) and greater capillary density (338.5 +/- 14.7 versus 259.8 +/- 9.2 vessels per mm2; n = 6-8, P < 0.001) than MIC hearts. Direct EPO injection reduced post-MI myocardial apoptosis by approximately 41% (0.27 +/- 0.03% versus 0.42 +/- 0.03%; n = 6, P= 0.005). The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit(+) and CD34(+) stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood (n = 7, P < 0.05) and 48 hrs in the infarcted hearts (n = 6, P < 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts (n = 7, P < 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c-Kit(+) and CD34(+) stem cells via the enhanced expression of chemoattractant SDF-1.

Localized and sustained SDF-1 gene release mediated by fibronectin films: A potential method for recruiting stem cells.

Gene-activated matrix has wide potential utilization in tissue engineering. It may genetically modify cells with plasmid DNA encoding therapeutic genes and allow sustained expression and release of the proteins to surrounding tissues. In this study, we assessed the feasibility of the local gene release from human fibronectin (HFN) substrate and the efficacy of local release of stromal cell-derived factor-1 (SDF-1) gene on c-kit+ cell homing. Cationic polymer polyethylenimine (25kDa PEI) was used as non-viral DNA vector. Gene-activated HFN (GAH) was prepared by mixing PEI/DNA complexes with HFN substrate. The DNA retardation, the complex size, and the DNA release speed from the GAH were studied. The in vitro transfection was optimized by luciferase expression and cell viability assay in the COS7 cell line. Localized gene expression in COS7 cells cultured on the GAH was assessed by LacZ and GFP-N3-SDF-1 marker genes. Ckit+ cell homing was investigated in response to the local in vitro SDF-1 expression from rat mesenchymal stem cells (RMSCs) cultured on GAH. Results showed GAH allows long time-sustained DNA release, localized gene delivery, and high transfection efficiency. Local SDF-1 expression with GAH is a promising method to induce targetable stem cell homing.

Single high-dose intramyocardial administration of erythropoietin promotes early intracardiac proliferation, proves safety and restores cardiac performance after myocardial infarction in rats.

Various studies demonstrate erythropoietin (EPO) as a cardioprotective growth hormone. Recent findings reveal EPO in addition might induce proliferation cascades inside myocardium. We aimed to evaluate whether a single high-dose intramyocardial EPO administration safely elevates early intracardiac cell proliferation after myocardial infarction (MI). Following permanent MI in rats EPO (3000 U/kg) in MI EPO-treatment group (n=99) or saline in MI control group (n=95) was injected along the infarction border. Intramyocardial EPO injection activated the genes of cyclin D1 and cell division cycle 2 kinase (cdc2) at 24 h after MI (n=6, P<0.05) evaluated by real time-PCR. The number of Ki-67+ intracardiac cells analyzed following immunohistochemistry was significantly enhanced by 45% in the peri-infarction zone at 48 h after EPO treatment (n=6, P<0.001). Capillary density was significantly enhanced by 17% as early as seven days (n=6, P<0.001). After six weeks, left ventricular performance assessed by conductance catheters was restored under baseline and dobutamine induced stress conditions (n=11-14, P<0.05). No thrombus formation was observed in the heart and in distant organs. No deleterious systemic adverse effects were apparent. Single high-dose intramyocardial EPO delivery proved safety and promoted early intracardiac cell proliferation, which might in part have contributed to an attenuated myocardial functional decline.

Deforestation and the structure of frog communities in the Humedale Terraba-Sierpe, Costa Rica.

Loss of tropical forests is a major cause of biodiversity loss worldwide. Although drastic modification of the habitat has been shown to negatively affect amphibians, we are far from a complete understanding of the response of amphibian communities to deforestation. We studied frog assemblages in a gradient of forest modification in a humid area of Costa Rica, where the primary forest has been partially converted into pasture. The study area is a mosaic of primary palm forest, abandoned pasture covered by secondary forest, and pasture. Species richness was assessed by randomized walk surveys and audio strip transects. We also measured ecological features to evaluate the relationship between landscape alteration and amphibian distribution. The study area hosted a large number of amphibian species. We focused our monitoring on six anurans: Leptodactylus labialis, Eleutherodactylus fitzingeri, E. diastema, Hyla rosenbergi, H. microcephale, and Cochranella granulosa. Three species (L. labialis, H. rosenbergi, and H. microcephala) were most abundant in pasture areas with livestock presence, while E. fitzingeri, E. diastema, and C. granulosa were associated with primary forest. Most of the variation in community structure was explained by the joint effect of forest alteration and presence of livestock. Whereas forest specialists suffer direct negative effect from deforestation, generalist species can take advantage of forest alteration and the presence of farm animals. Species that are able to take advantage of the new environmental characteristics associated with human modifications of landscapes will come to prevail in the new communities.

Is the intravascular administration of mesenchymal stem cells safe? Mesenchymal stem cells and intravital microscopy.

We investigated the kinetics of human mesenchymal stem cells (MSCs) after intravascular administration into SCID mouse cremaster vasculature by intravital microscopy. MSCs were injected into abdominal aorta through left femoral artery at two different concentrations (1 x 10(6) or 0.2 x 10(6) cell). Arterial blood velocity decrease by 60 and 18% 1 min after high/low dose MSCs injection respectively. The blood microcirculation was interrupted after 174+/-71 and 485+/-81 s. Intravital microscopy observation and histopathologic analysis of cremaster muscles indicated MSCs were entrapped in capillaries in both groups. 40 and 25% animals died of pulmonary embolism respectively in both high and low MSCs dose groups, which was detected by histopathologic analysis of the lungs. Intraarterial MSCs administration may lead to occlusion in the distal vasculature due to their relatively large cell size. Pulmonary sequestration may cause death in small laboratory animals. MSCs should be used cautiously for intravascular transplantation.

Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

Bcl-2 engineered MSCs inhibited apoptosis and improved heart function.

Engraftment of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for postinfarction left ventricular dysfunction. However, limited cell viability after transplantation into the myocardium has restricted its regenerative capacity. In this study, we genetically modified MSCs with an antiapoptotic Bcl-2 gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a rat left anterior descending ligation model via intracardiac injection. Rat MSCs were manipulated to overexpress the Bcl-2 gene. In vitro, the antiapoptotic and paracrine effects were assessed under hypoxic conditions. In vivo, the Bcl-2 gene-modified MSCs (Bcl-2-MSCs) were injected after myocardial infarction. The surviving cells were tracked after transplantation. Capillary density was quantified after 3 weeks. The left ventricular function was evaluated by pressure-volume loops. The Bcl-2 gene protected MSCs against apoptosis. In vitro, Bcl-2 overexpression reduced MSC apoptosis by 32% and enhanced vascular endothelial growth factor secretion by more than 60% under hypoxic conditions. Transplantation with Bcl-2-MSCs increased 2.2-fold, 1.9-fold, and 1.2-fold of the cellular survival at 4 days, 3 weeks, and 6 weeks, respectively, compared with the vector-MSC group. Capillary density in the infarct border zone was 15% higher in Bcl-2-MSC transplanted animals than in vector-MSC treated animals. Furthermore, Bcl-2-MSC transplanted animals had 17% smaller infarct size than vector-MSC treated animals and exhibited functional recovery remarkably. Our current findings support the premise that transplantation of antiapoptotic gene-modified MSCs may have values for mediating substantial functional recovery after acute myocardial infarction.