Adam Oskowitz

Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression

We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF.

Serum-deprived human multipotent mesenchymal stromal cells (MSCs) are highly angiogenic.

Recent reports have indicated that mesenchymal stromal cells (MSCs) from bone marrow have a potential in vascular remodeling and angiogenesis. Here, we report a unique phenomenon that under serum-deprived conditions MSCs survive and replicate. Secretome analysis of MSCs grown under serum-deprived conditions (SD-MSCs) identified a significant upregulation of prosurvival and angiogenic factors including VEGF-A, ANGPTs, IGF-1, and HGF. An ex vivo rat aortic assay demonstrated longer neovascular sprouts generated from rat aortic rings cultured in SD-MSC-conditioned media compared to neovascular sprouts from aortas grown in MSC-conditioned media. With prolonged serum deprivation, a subpopulation of SD-MSCs began to exhibit an endothelial phenotype. This population expressed endothelial-specific proteins including VEGFR2, Tie2/TEK, PECAM/CD31, and eNOS and also demonstrated the ability to uptake acetylated LDL. SD-MSCs also exhibited enhanced microtubule formation in an in vitro angiogenesis assay. Modified chick chorioallantoic membrane (CAM) angiogenesis assays showed significantly higher angiogenic potential for SD-MSCs compared to MSCs. Analysis of CAMs grown with SD-MSCs identified human-specific CD31-positive cells in vascular structures. We conclude that under the stress of serum deprivation MSCs are highly angiogenic and a population of these cells has the potential to differentiate into endothelial-like cells.

Activation of autophagy in mesenchymal stem cells provides tumor stromal support

Recent studies have implicated multipotential mesenchymal stem cells (MSCs) as an aid to breast cancer cell proliferation and metastasis, partly as a result of the MSCs secretome. As the tumor gets beyond 2 mm in diameter, the stromal cells could undergo starvation due to the lack of sufficient nutrients in solid tumor microenvironment. In this study, we investigated the survival mechanisms used by stressed stromal cells in breast cancers. We used serum-deprived mesenchymal stem cells (SD-MSCs) and MCF-7 breast cancer cells as model system with a hypothesis that stromal cells in the nutrient-deprived core utilize survival mechanisms for supporting surrounding cells. We tested this hypothesis using in vivo tumor xenografts in immunodeficient mice, which indicated that SD-MSCs supported MCF-7 tumor growth by protection from apoptosis. Histochemical assays showed that SD-MSCs-injected tumors exhibited higher cellularity, decreased apoptosis and decreased differentiation. Beclin-1 staining indicated autophagic areas surrounded by actively proliferating cells. Furthermore, in vitro studies demonstrate that SD-MSCs survive using autophagy and secrete paracrine factors that support tumor cells following nutrient/serum deprivation. Western blot and immunocytochemistry analysis of SD-MSCs demonstrated upregulation and perinuclear relocation of autophagy key regulators such as beclin-1, ATG10, ATG12, MAP-LC3 and lysosomes. Electron microscopic analysis detected a time-dependent increase in autophagosome formation and HDAC6 activity assays indicated the upregulation of autophagy. Taken together, these data suggest that under nutrient-deprived conditions that can occur in solid tumors, stromal cells utilize autophagy for survival and also secrete anti-apoptotic factors that can facilitate solid tumor survival and growth.

Epigenetic reprogramming of IGF1 and leptin genes by serum deprivation in multipotential mesenchymal stromal cells.

Recent studies on the therapeutic effect of multipotential mesenchymal stem cells (MSCs) in various models of injury have shown that paracrine factors secreted by MSCs are responsible for tissue repair with very little engraftment. In this study we tested the hypothesis that MSCs under stress undergo epigenetic modifications that direct secretion of paracrine factors responsible for tissue repair. Microarray assays of MSCs that had been deprived of serum (SD‐MSCs), to induce stress, demonstrated an increase in the expression of several angiogenic, prosurvival, and antiapoptotic factors, including insulin‐like growth factor 1 (IGF1) and leptin. Real‐time polymerase chain reaction assays demonstrated a >200‐fold increase in the expression of IGF1 and leptin in SD‐MSCs. Chromatin immunoprecipitation of SD‐MSCs revealed histone tail modifications consistent with transcriptional activation of IGF1 and leptin promoters in a reversible manner. To identify the functional significance of the epigenetic changes in stressed MSCs, we tested the prosurvival properties of SD‐MSCs and the ability of conditioned medium from SD‐MSCs to enhance survival of apoptotic cancer cells. First, we showed that SD‐MSCs are more resistant to oxidative damage than MSCs using alkaline comet assays. Next, we demonstrated that conditioned medium from SD‐MSCs decreased staurosporin‐induced cell death in the KHOS osteosarcoma cell line, and that this effect was partially reversed by immunodepletion of IGF1 or leptin from the conditioned medium. In conclusion, we demonstrate that serum deprivation induces epigenetic changes in MSCs to upregulate the expression of the proangiogenic and antiapoptotic factors IGF1 and leptin. STEM CELLS 2009;27:375–382

Drosha regulates hMSCs cell cycle progression through a miRNA independent mechanism.

Recently we demonstrated that the miRNA regulate human mesenchymal stem cells (hMSCs) differentiation. To determine the role of the miRNA pathway in hMSCs proliferation, Drosha and Dicer knockdown hMSCs were generated using a lentiviral based tetracycline inducible shRNA. hMSCs with reduced Drosha expression had a significantly reduced proliferation rate, while hMSCs with reduced Dicer expression displayed a proliferation rate similar to untransduced cells. Cell cycle analysis identified that unlike Dicer knockdown, Drosha knockdown hMSCs contained an increased number of G1 phase cells, with a reduced level of cells in S phase, compared to controls. ELISAs of hMSCs revealed decreased levels of pRB and stable levels of total RB with Drosha knockdown. Two key regulators of the G1/S phase transition, cyclin dependent kinase inhibitor 2A (p16) and cyclin dependent kinase inhibitor 2B (p15), were increased in Drosha knockdown cells but not in Dicer knockdown. Transcripts of 28S and 18S rRNA were significantly reduced in Drosha knockdown hMSCs, with no change in rRNA levels in Dicer knockdown hMSCs. 45S pre-rRNA transcripts were not significantly different in either knockdown model. The above results indicate that Drosha modifies hMSCs proliferation through a miRNA independent mechanism, potentially by regulating rRNA processing.

Contemporary outcomes after venography-guided treatment of patients with May-Thurner syndrome.

OBJECTIVE Patients with May-Thurner syndrome (MTS) present with a spectrum of findings ranging from mild left leg edema to extensive iliofemoral deep venous thrombosis (DVT). Whereas asymptomatic left common iliac vein (LCIV) compression can be seen in a high proportion of normal individuals on axial imaging, the percentage of these persons with symptomatic compression is small, and debate exists about the optimal clinical and diagnostic criteria to treat these lesions in patients with symptomatic venous disease. We evaluated our approach to venography-guided therapy for individuals with symptomatic LCIV compression and report the outcomes. METHODS All patients with suspected May-Thurner compression of the LCIV between 2008 and 2015 were analyzed retrospectively. Patients with chronic iliocaval lesions not associated with compression of the LCIV were excluded from analysis. Criteria for intervention included LCIV compression in the setting of (1) leg edema/venous claudication with associated venographic findings (collateralization, iliac contrast stagnation, and contralateral cross cross-filling), or (2) left leg deep venous thrombosis. Outcome measures included presenting Clinical, Etiology, Anatomy, Pathophysiology (CEAP) score, postintervention CEAP score, primary patency, and secondary patency. Technical success was defined as successful stent implantation without intraoperative device complications, establishment of in-line central venous flow, and less than 30% residual LCIV stenosis. RESULTS Of the 63 patients evaluated, 32 (51%) had nonthrombotic MTS and presented with leg edema (100%) or venous claudication (47%). Thirty-one patients (49%) had thrombotic MTS and presented with acute (26%) or chronic (71%) DVT, leg edema (100%), or venous claudication (74%). The mean presenting CEAP score was 3.06 and 3.23 for nonthrombotic and thrombotic MTS, respectively. Forty-four patients (70%) underwent successful intervention with primary stenting (70%) or thrombolysis and stenting (30%); 14 nonthrombotic MTS patients were treated conservatively with compression therapy alone, and 5 thrombotic MTS patients were treated with lysis or angioplasty alone. Clinical improvement and decrease in CEAP score occurred in 95% and 77% of stented patients compared with 58% and 32% of nonstented patients. Complete symptom resolution was achieved in 48% of patients overall, or 64% of stented patients and only 21% of nonstented patients. Complications included two early reocclusions. Primary and secondary 2-year patency rates were 93% and 97% (mean follow-up, 20.3 months) for stented patients. CONCLUSIONS Venography-guided treatment of MTS is associated with excellent 1-year patency rates and a significant reduction in symptoms and CEAP score. Treating symptomatic MTS patients on the basis of physiologically relevant venographic findings rather than by intravascular ultrasound imaging alone results in excellent long-term patency and clinical outcomes but may result in undertreatment of some patients who could benefit from stent implantation.

Infrarenal aortic repair with or without false lumen intentional placement of endografts for hybrid management of complex aortic dissection

Objective: A D-dimer (DD) level in addition to the pretest clinical probability (PCP) score using Wells criteria can safely exclude deep venous thrombosis (DVT). In the current study, we sought to examine the correlation between the proposed DD level and the PCP model in approaching diagnosis and to determine the location and severity of a possible DVT in high-volume hospitals. Methods: Patientswhopresentedwith limbswellingwithorwithoutpain were retrospectively examined from June 2014 to July 2015. After calculation of PCP, patients were divided into low, moderate, and high risk. Electronic DD results using enzyme-linked immunosorbent assay, PCP data, and venous duplex ultrasound (VDU) analysis data were merged and analyzed on the basis of receiver operating characteristic curve to determine the DD cutoff point for each PCP score (Fig). An abnormal DD level with an average value of

CREB phosphorylation and c‐Fos expression in the hippocampus of rats during acquisition and recall of a socially transmitted food preference

0.6mg/Lwas correlated to positive DVT to differentiate proximal (above popliteal vein) from distal (below popliteal vein). Results: Data of 1909 patients were analyzed, and 239 (12.5%) were excluded secondary to previous history of DVT. Average age was 62.1 6 16.3 years, and 55.7% were female; the majority presented with limb pain and edema (87%). DD analysis was completed in 202 patients and correlated with all positive and negative DVT results (100% sensitivity and positive predictive value). In 26 of 202, DD levels were in the normal range (0.1-0.59 mg/L), all of which were negative for DVT (100% sensitive); in 51 of 202 with DD values in the range of 0.6 to 1.2 mg/L, only 3 DVTs were recorded, all of them distal. Also, for 668 patients with PCP score <1, patients with positive DD results were sent for VDU examination. Thus, 745 patients had an un-needed VDU study, a potential charge savings for VDU for all (745

Human mesenchymal stromal cells (MSCs) reduce neointimal hyperplasia in a mouse model of flow-restriction by transient suppression of anti-inflammatory cytokines.

1557 1⁄4

Hybrid treatment of aortic arch aneurysms.

1,159,965) and DD for all (745