Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopolesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells

Low dose cranial irradiation-induced cerebrovascular damage is reversible in mice

BACKGROUND: High-dose radiation-induced blood-brain barrier breakdown contributes to acute radiation toxicity syndrome and delayed brain injury, but there are few data on the effects of low dose cranial irradiation. Our goal was to measure blood-brain barrier changes after low (0.1 Gy), moderate (2 Gy) and high (10 Gy) dose irradiation under in vivo and in vitro conditions. METHODOLOGY: Cranial irradiation was performed on 10-day-old and 10-week-old mice. Blood-brain barrier permeability for Evans blue, body weight and number of peripheral mononuclear and circulating endothelial progenitor cells were evaluated 1, 4 and 26 weeks postirradiation. Barrier properties of primary mouse brain endothelial cells co-cultured with glial cells were determined by measurement of resistance and permeability for marker molecules and staining for interendothelial junctions. Endothelial senescence was determined by senescence associated β-galactosidase staining. PRINCIPLE FINDINGS: Extravasation of Evans blue increased in cerebrum and cerebellum in adult mice 1 week and in infant mice 4 weeks postirradiation at all treatment doses. Head irradiation with 10 Gy decreased body weight. The number of circulating endothelial progenitor cells in blood was decreased 1 day after irradiation with 0.1 and 2 Gy. Increase in the permeability of cultured brain endothelial monolayers for fluorescein and albumin was time- and radiation dose dependent and accompanied by changes in junctional immunostaining for claudin-5, ZO-1 and β-catenin. The number of cultured brain endothelial and glial cells decreased from third day of postirradiation and senescence in endothelial cells increased at 2 and 10 Gy. CONCLUSION: Not only high but low and moderate doses of cranial irradiation increase permeability of cerebral vessels in mice, but this effect is reversible by 6 months. In-vitro experiments suggest that irradiation changes junctional morphology, decreases cell number and causes senescence in brain endothelial cells

Regeneration of Soft Tissues Is Promoted by MMP1 Treatment after Digit Amputation in Mice

The ratio of matrix metalloproteinases (MMPs) to the tissue inhibitors of metalloproteinases (TIMPs) in wounded tissues strictly control the protease activity of MMPs, and therefore regulate the progress of wound closure, tissue regeneration and scar formation. Some amphibians (i.e. axolotl/newt) demonstrate complete regeneration of missing or wounded digits and even limbs; MMPs play a critical role during amphibian regeneration. Conversely, mammalian wound healing re-establishes tissue integrity, but at the expense of scar tissue formation. The differences between amphibian regeneration and mammalian wound healing can be attributed to the greater ratio of MMPs to TIMPs in amphibian tissue. Previous studies have demonstrated the ability of MMP1 to effectively promote skeletal muscle regeneration by favoring extracellular matrix (ECM) remodeling to enhance cell proliferation and migration. In this study, MMP1 was administered to the digits amputated at the mid-second phalanx of adult mice to observe its effect on digit regeneration. Results indicated that the regeneration of soft tissue and the rate of wound closure were significantly improved by MMP1 administration, but the elongation of the skeletal tissue was insignificantly affected. During digit regeneration, more mutipotent progenitor cells, capillary vasculature and neuromuscular-related tissues were observed in MMP1 treated tissues; moreover, there was less fibrotic tissue formed in treated digits. In summary, MMP1 was found to be effective in promoting wound healing in amputated digits of adult mice. © 2013 Mu et al

The Immature Heart: The Roles of Bone Marrow Stromal Stem Cells in Growth and Myocardial Repair

Studies have shown that adult bone marrow derived stem cells (MSCs) can participate in repair of myocardial injury in adult hearts, as well as in cardiac growth during fetal development in utero. Yet, no studies have evaluated the role of MSCs with respect to normal growth or tissue repair in immature hearts after birth. The present study examines whether MSCs may participate in the myocardial growth and injury in the post-natal immature hearts. MSCs were isolated from adult Lewis rats and labeled with Lac-Z gene using retroviral vectors. These MSCs were injected systemically into groups of neonatal (NB=2days-old), immature (B=30days-old) and adult (A=>3months-old) isogeneic Lewis rats. Additionally, left coronary artery ligation was carried out in subgroups of immature (BL) and adult (AL) rats one week after MSCs injection. The hearts were harvested serially from 2-days to 6-weeks, stained with X-Gal for labeled MSCs. Cardiomyocyte phenotypic expression was evaluated by immunohistological staining for Troponin I-C and Connexin-43. Labeled MSCs were found to home into the bone marrow in all rats of different developmental stages. They could be recruited from bone marrow into the infarcted site of myocardium only in groups AL and BL. They were also capable of differentiating into cardiomyocyte phenotype after myocardial injury. In contrast to that reported in the developing fetus, MSCs did not appear to contribute to the growth of non-injured hearts after birth. However, they can be recruited from the bone marrow and regenerate damaged myocardium both in the adult and in the immature hearts

Components of the Hematopoietic Compartments in Tumor Stroma and Tumor-Bearing Mice

Solid tumors are composed of cancerous cells and non-cancerous stroma. A better understanding of the tumor stroma could lead to new therapeutic applications. However, the exact compositions and functions of the tumor stroma are still largely unknown. Here, using a Lewis lung carcinoma implantation mouse model, we examined the hematopoietic compartments in tumor stroma and tumor-bearing mice. Different lineages of differentiated hematopoietic cells existed in tumor stroma with the percentage of myeloid cells increasing and the percentage of lymphoid and erythroid cells decreasing over time. Using bone marrow reconstitution analysis, we showed that the tumor stroma also contained functional hematopoietic stem cells. All hematopoietic cells in the tumor stroma originated from bone marrow. In the bone marrow and peripheral blood of tumor-bearing mice, myeloid populations increased and lymphoid and erythroid populations decreased and numbers of hematopoietic stem cells markedly increased with time. To investigate the function of hematopoietic cells in tumor stroma, we co-implanted various types of hematopoietic cells with cancer cells. We found that total hematopoietic cells in the tumor stroma promoted tumor development. Furthermore, the growth of the primary implanted Lewis lung carcinomas and their metastasis were significantly decreased in mice reconstituted with IGF type I receptor-deficient hematopoietic stem cells, indicating that IGF signaling in the hematopoietic tumor stroma supports tumor outgrowth. These results reveal that hematopoietic cells in the tumor stroma regulate tumor development and that tumor progression significantly alters the host hematopoietic compartment

Identification of TNF-α and MMP-9 as potential baseline predictive serum markers of sunitinib activity in patients with renal cell carcinoma using a human cytokine array

BACKGROUND: Several drugs are available to treat metastatic renal-cell carcinoma (MRCC), and predictive markers to identify the most adequate treatment for each patient are needed. Our objective was to identify potential predictive markers of sunitinib activity in MRCC. METHODS: We collected sequential serum samples from 31 patients treated with sunitinib. Sera of six patients with extreme phenotypes of either marked responses or clear progressions were analysed with a Human Cytokine Array which evaluates 174 cytokines before and after treatment. Variations in cytokine signal intensity were compared between both groups and the most relevant cytokines were assessed by ELISA in all the patients. RESULTS: Twenty-seven of the 174 cytokines varied significantly between both groups. Five of them (TNF-alpha, MMP-9, ICAM-1, BDNF and SDF-1) were assessed by ELISA in 21 evaluable patients. TNF-alpha and MMP-9 baseline levels were significantly increased in non-responders and significantly associated with reduced overall survival and time-to-progression, respectively. The area under the ROC curves for TNF-alpha and MMP-9 as predictive markers of sunitinib activity were 0.83 and 0.77. CONCLUSION: Baseline levels of TNF-alpha and MMP-9 warrant further study as predictive markers of sunitinib activity in MRCC. Selection of patients with extreme phenotypes seems a valid method to identify potential predictive factors of response

Mechanism of endothelial progenitor cell recruitment into neo-vessels in adjacent non-tumor tissues in hepatocellular carcinoma

Abstract Background We investigated the distribution and clinical significance of mobilized endothelial progenitor cells (EPCs) in hepatocellular carcinoma (HCC). We found that many more EPCs were recruited to nonmalignant liver tissue (especially into adjacent non-tumor tissues (AT)) than to tumor vessels. These results suggest that the mechanism underlying the recruitment of EPCs into microvessels in AT merits further investigation Methods Angiogenic factors were detected in three tissue microarrays comprising normal liver, paired tumor tissue (TT) and AT from 105 patients (who had undergone hepatectomy for HCC) using immunohistochemistry. Also, the number of EPCs (positive for Sca-1, Flk-1 and c-Kit) in the blood and liver of cirrhotic mice were determined by flow cytometry and immunohistochemistry. The distribution of these labeled EPCs in tumor and non-tumor tissues was then studied. Results The results from the tissue microarrays showed that the expression levels of VEGF-A, bFGF, TGF-β, MCP-1, TSP-1, MMP-9, TIMP-2, and endostatin were significantly higher in AT than in either normal liver or TT (p Conclusions Both liver cirrhosis and HCC led to increased expression of pro-angiogenic factors, which resulted in the recruitment of EPCs into AT. Also, EPCs were mobilized, recruited and homed to cirrhotic liver. The unique pathology of HCC coupled with liver cirrhosis may, therefore, be associated with the distribution and function of EPCs.</p

Cerebrospinal fluid matrix metalloproteinase-9 increases during treatment of recurrent malignant gliomas

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinases (MMPs) are enzymes that promote tumor invasion and angiogenesis by enzymatically remodeling the extracellular matrix. MMP-2 and MMP-9 are the most abundant forms of MMPs in malignant gliomas, while a 130 kDa MMP is thought to be MMP-9 complexed to other proteinases. This study determined whether doxycycline can block MMP activity <it>in vitro</it>. We also measured MMP-2 and MMP-9 levels in cerebrospinal fluid (CSF) from patients with recurrent malignant gliomas.</p> <p>Methods</p> <p>To determine whether doxycycline can block MMP activity, we measured the extent of doxycyline-mediated MMP-2 and MMP-9 inhibition <it>in vitro </it>using epidermal growth factor receptor (EGFR) transfected U251 glioma cell lines. MMP activity was measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography. In addition, patients underwent lumbar puncture for CSF sampling at baseline, after 6 weeks (1 cycle), and after 12 weeks (2 cycles), while being treated with a novel chemotherapy regimen of irinotecan, thalidomide, and doxycycline designed to block growth/proliferation, angiogenesis, and invasion. Irinotecan was given at 125 mg/m²/week for 4 weeks in 6-week cycles, together with continuous doxycycline at 100 mg twice daily on Day 1 and 50 mg twice daily thereafter. Daily thalidomide dose in our cohort was 400 mg. Tumor progression was monitored by magnetic resonance imaging (MRI).</p> <p>Results</p> <p>Doxycyline <it>in vitro </it>completely abolished MMP-9 activity at 500 μg/ml while there was only 30 to 50% inhibition of MMP-2 activity. Four patients respectively completed 4, 3, 1, and 2 cycles of irinotecan, thalidomide, and doxycycline. Patient enrollment was terminated after one patient developed radiologically defined pulmonary embolism, and another had probable pulmonary embolism. Although CSF MMP-2 and 130 kDa MMP levels were stable, MMP-9 level progressively increased during treatment despite stable MRI.</p> <p>Conclusion</p> <p>Doxycycline can block MMP-2 and MMP-9 activities from glioma cells <it>in vitro</it>. Increased CSF MMP-9 activity could be a biomarker of disease activity in patients with malignant gliomas, before any changes are detectable on MRI.</p

c-Kit-Mediated Functional Positioning of Stem Cells to Their Niches Is Essential for Maintenance and Regeneration of Adult Hematopoiesis

The mechanism by which hematopoietic stem and progenitor cells (HSPCs) through interaction with their niches maintain and reconstitute adult hematopoietic cells is unknown. To functionally and genetically track localization of HSPCs with their niches, we employed novel mutant loxPs, lox66 and lox71 and Cre-recombinase technology to conditionally delete c-Kit in adult mice, while simultaneously enabling GFP expression in the c-Kit-deficient cells. Conditional deletion of c-Kit resulted in hematopoietic failure and splenic atrophy both at steady state and after marrow ablation leading to the demise of the treated adult mice. Within the marrow, the c-Kit-expressing GFP+ cells were positioned to Kit ligand (KL)-expressing niche cells. This c-Kit-mediated cellular adhesion was essential for long-term maintenance and expansion of HSPCs. These results lay the foundation for delivering KL within specific niches to maintain and restore hematopoiesis

Circulating CD133+CD34+ progenitor cells inversely correlate with soluble ICAM-1 in early ischemic stroke patients

<p>Abstract</p> <p>Background and Purpose</p> <p>Both endothelial progenitor cells (EPC) and markers of neuroinflammation are candidate biomarkers for stroke severity and outcome prediction. A relationship between EPC and neuroinflammatory markers in early stroke is not fully elucidated. The objectives were to investigate correlations between EPC and neuroinflammation markers (adhesion molecules ICAM-1, VCAM-1, E-selectin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, endothelin (ET)-1, markers of tissue injury (matrix metalloproteinases (MMP)-9 and tissue inhibitor of matrix metalloproteinases (TIMP)-1) in early stroke patients.</p> <p>Methods</p> <p>We prospectively recruited symptomatic patients with ischemic cerebrovascular disease. We assessed stroke severity by using of acute (diffusion-weighted imaging (DWI) and final lesion volumes (fluid attenuated inversion recovery (FLAIR). We measured serum soluble ICAM-1, VCAM-1, E-selectin, MMP-9, TIMP-1 and plasma TNF-α, IL-6, ET-1 by ELISA, and quantified EPC in mononuclear fraction of peripheral blood on days 1 and 3 in 17 patients (mean(SD) age 62(14), with admission National Institutes of Health Stroke Scale (NIHSS) 10(8)) selected from 175 patients with imaging confirmed ischemic stroke. Non-parametric statistics, univariate and multivariate analysis were used.</p> <p>Results</p> <p>Only ICAM-1 inversely correlated with EPC subset CD133+CD34+ on day 1 (Spearman r = -0.6, p < 0.01) and on day 3 (r = -0.967, p < 0.001). This correlation remained significant after adjustment for age and NIHSS (beta -0.992, p < 0.004), for glucose and systolic blood pressure (beta -0.86, p < 0.005), and for white blood cells and hematocrit (beta -1.057, p < 0.0001) on day 3. MMP-9 (r = 0.509, p < 0.04) and MMP-9/TIMP-1 (r = 0.59, p < 0.013) on day 1 correlated with acute lesion volume. Both IL-6 (r = 0.624, p < 0.01) and MMP-9/TIMP-1 (r = 0.56, p < 0.02) correlated with admission NIHSS.</p> <p>Conclusion</p> <p>Our study showed that high ICAM-1 is associated with low CD133+CD34+subset of EPC. Biomarkers of neuroinflammation may predict tissue injury and stroke severity in early ischemia.</p