Amnion-derived mesenchymal stem cells improve viability of endothelial cells exposed to shear stress in ePTFE grafts.

PURPOSE:: Blood vessel reconstruction is an increasing need of patients suffering from cardiovascular diseases. For the development of microvascular prostheses, efficient endothelialization is mandatory to prevent graft occlusion. Here, we assessed the impact of amnion-derived mesenchymal stem/stromal cells (hAMSC), known for their important angiogenic potential, on the integrity and stability of endothelial cells exposed to shear stress in vascular grafts. METHODS:: Human placental endothelial cells (hPEC) were cultured at the inner surface of an expanded polytetrafluoroethylene (ePTFE) graft positioned within a bioreactor and exposed to a minimal shear stress of 0.015 dyne/cm2 or a physiological shear stress of 0.92 dyne/cm2. hAMSC attached to the outer graft surface were able to interact with human placental endothelial cells by paracrine factors. RESULTS:: Microscopical analysis and evaluation of glucose/lactate metabolism evidenced successful cell seeding of the graft: hPEC formed a stable monolayer, hAMSC showed a continuous growth during 72 h incubation. hAMSC improved the viability of hPEC exposed to 0.015 dyne/cm2 as shown by a decreased lactate dehydrogenase release of 13% after 72 h compared to hPEC single culture. The viability-enhancing effect of hAMSC on hPEC was further improved by 13% under physiological shear stress. Angiogenesis array analysis revealed that hPEC exposed to physiological shear stress and hAMSC co-culture reduced the secretion of angiogenin, GRO, MCP-1, and TIMP-2. CONCLUSION:: hAMSC exerted best survival-enhancing effects on hPEC under exposure to physiological shear stress and modulated endothelial function by paracrine factors. Our data support further studies on the development of grafts functionalized with hAMSC-derived secretomes to enable fast clinical application.

Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds.

OBJECTIVE: Mesenchymal stem/stromal cells derived from human term placentas (PMSCs) are novel therapeutic agents and more topical than ever. Here we evaluated the effects of three types of PMSCs on wound healing in an in vivo mouse model: Amnion-derived MSCs (AMSCs), blood vessel-derived MSCs (BV-MSCs) from the chorionic plate and Wharton's jelly-derived MSCs (WJ-MSCs) from the umbilical cord. METHODS: We topically applied PMSCs onto skin wounds in mice using the dermal substitute Matriderm® as carrier and evaluated wound healing parameters. In addition, we investigated the effects of all PMSC types under co-application with placental endothelial cells (PLECs). After 8 days, we compared the percent of wound closure and the angiogenic potential between all groups. RESULTS: AMSCs, BV-MSCs and WJ-MSCs significantly induced a faster healing and a higher number of blood vessels in the wound when compared to controls (Matriderm®-alone). PLECs did not further improve the advantageous effects of PMSC-treatment. Quantitative data and 3D analysis by high resolution episcopic microscopy confirmed a lower density of vessels in Matriderm®/PMSCs/PLECs co-application compared to Matriderm®/PMSCs treatment. CONCLUSION: Results indicate that all three PMSC types exert similar beneficial effects on wound closure and neovascularization in our mouse model. PRACTICE: Using Matriderm® as carrier for PMSCs propagates rapid cell migration towards the wound area that allows a fast and clinically practicable method for stem cell application. IMPLICATIONS: These promising effects warrant further investigation in clinical trials.

miR-146a, miR-146b, and miR-155 increase expression of IL-6 and IL-8 and support HSP10 in an In vitro sepsis model.

microRNAs (miRNAs) play an essential role in inflammation processes including sepsis. This study aimed to identify miRNAs as candidates for therapies that are involved in the innate immune response and to assess their potential functions in the activation of the endothelium. We stimulated THP-1 monocytes with 10 ng/ml LPS for 4 h and used the supernatant for the stimulation of human umbilical vein endothelial cells (HUVEC) or human pulmonary microvascular endothelial cells (HPMEC) for 16 h. miRNA array analysis (of 1,891 miRNAs) identified a 1.5-fold upregulation of miR-146a, miR-146b, and miR-155 in stimulated endothelial cells. HUVEC were transfected with miRNA inhibitors for miR-146a, miR-146b, and miR-155 to investigate the function of these miRNAs in endothelial inflammatory pathways. Inhibition of miR-146a resulted in a diminished release of interleukin (IL)-6 and IL-8 by respective 68% and 55% (P<0.001). Inhibition of miR-146b reduced the expression of IL-6 by 49% (P<0.001). Inhibition of miR-155 reduced the expression of IL-6 and IL-8 by respective 31% (P<0.001) and 14%. The inhibition of miR-146a, miR-146b, and miR-155 reduced the release of HSP10 by 50%, 35%, and 69% (P<0.05), respectively, but did not influence the expression of HSP27 or TXA2. In conclusion, miR-146a, miR-146b, and miR-155 are exerting anti-inflammatory properties by down-regulating IL-6 and IL-8, and influencing the expression of HSP10 in the activated endothelium. We provide evidence for the central role of selected miRNAs in sepsis and their use in the development of small interfering RNA therapeutics to target immune cells and sepsis pathways.

Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses.

Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo These 3D models displayed augmented anti-tumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies.

Comparison of Matrigel and Matriderm as a carrier for human amnion-derived mesenchymal stem cells in wound healing.

Amnion-derived mesenchymal stem cells (AMSC) are a promising tool in regenerative medicine. Here we evaluated the utility of Matrigel and Matriderm as carrier for the topical application of AMSC to mice skin wounds. In both application forms, AMSC promoted neovascularization of the wound area. Matrigel proved as excellent matrix for AMSC and immigrating mouse cells, but the solid Matriderm enabled a more adequate positioning of AMSC into the wound. Although AMSC did not attach to Matriderm, they reliably induced wound reduction. Thus, a combined administration of AMSC/Matriderm could be beneficial to potentiate the encouraging effects on wound healing.

Atraumatic Pulsatile Leukocyte Circulation for Long-Term In Vitro Dynamic Culture and Adhesion Assays.

Low flow rate pumping of cell suspensions finds current applications in bioreactors for short-term dynamic cell culture and adhesion assays. The aim of this study was to develop an atraumatic pump and hemodynamically adapted test circuit to allow operating periods of at least several hours. A computer-controlled mini-pump (MP) was constructed based on non-occlusive local compression of an elastic tube with commercial bi-leaflet valves directing the pulsatile flow into a compliant circuit. Cell damage and activation in the system were tested with whole blood in comparison with a set with a conventional peristaltic pump (PP). Activation of circulating THP-1 monocytes was tested by measuring the expression of CD54 (ICAM-1). Additionally, monocyte-endothelial interactions were monitored using a parallel-plate flow chamber with an artificial stenosis. The system required a priming volume of only 20 mL, delivering a peak pulsatile flow of up to 35 mL/min. After 8 h, blood hemolysis was significantly lower for MP with 11 ± 3 mg/dL compared with PP with 100 ± 16 mg/dL. CD142 (tissue factor) expression on blood monocytes was 50% lower for MP. With MP, THP-1 cells could be pumped for extended periods (17 h), with no enhanced expression of CD54 permitting the long-term co-culture of THP-1 with endothelial cells and the analysis of flow pattern effects on cell adhesion. A low-damage assay setup was developed, which allows the pulsatile flow of THP-1 cells and investigation of their interaction with other cells or surfaces for extended periods of time.

Placental mesenchymal stromal cells derived from blood vessels or avascular tissues: what is the better choice to support endothelial cell function?

Mesenchymal stromal cells (MSCs) are promising tools for therapeutic revascularization of ischemic tissues and for support of vessel formation in engineered tissue constructs. Recently, we could show that avascular-derived MSCs from placental amnion release soluble factors that exhibit survival-enhancing effects on endothelial cells (ECs). We hypothesize that MSCs derived from placental blood vessels might have even more potent angiogenic effects. Therefore, we isolated and characterized MSCs from placental chorionic blood vessels (bv-MSCs) and tested their angiogenic potential in comparison to amnion-derived avascular MSCs (av-MSCs). bv-MSCs express a very similar surface marker profile compared with av-MSCs and could be differentiated toward the adipogenic and osteogenic lineages. bv-MSCs exert immunosuppressive properties on peripheral blood mononuclear cells, suggesting that they are suitable for cell transplantation settings. Conditioned medium (Cdm) from av-MSCs and bv-MSCs significantly enhanced EC viability, whereas only Cdm from bv-MSCs significantly increased EC migration and network formation (Matrigel assay). Angiogenesis array analysis of av- and bv-MSC-Cdm revealed a similar secretion pattern of angiogenic factors, including angiogenin, interleukins-6 and -8, and tissue inhibitors of matrix metalloproteinase-1 and 2. Enzyme-linked immunosorbent assay analysis showed that, in contrast to av-MSCs, bv-MSCs secreted vascular endothelial growth factor. In direct coculture with bv-MSCs, ECs showed a significantly increased formation of vessel-like structures compared with av-MSCs. With regard to therapeutic treatment, bv-MSCs and particularly their Cdm might be valuable to stimulate angiogenesis especially in ischemic tissues. av-MSCs and their Cdm could be beneficial in conditions when it is required to promote the survival and stabilization of blood vessels without the risk of unmeant angiogenesis.

The Resazurin Reduction Assay Can Distinguish Cytotoxic from Cytostatic Compounds in Spheroid Screening Assays.

Spheroid-based cellular screening approaches represent a highly physiologic experimental setup to identify novel anticancer drugs and an innovative preclinical model to reduce the high failure rate of anticancer compounds in clinical trials. The resazurin reduction (RR) assay, known as the alamarBlue or CellTiter-Blue assay, is frequently used to determine cell viability/proliferation capacity in eukaryotic cells. Whether this assay is applicable to assess viability in multicellular spheroids has not been evaluated. We analyzed the RR assay to measure cytotoxic and/or cytostatic responses in tumor cell spheroids compared with conventional 2D cultures. We found that tight cell-cell interactions in compact spheroids hamper resazurin uptake and its subsequent reduction to resorufin, leading to lowered reduction activity in relation to the actual cellular health/cell number. Treatment with staurosporine disrupted close cell-cell contacts, which increased resazurin reduction compared with untreated controls. Loss of tight junctions by trypsinization or addition of EGTA or EDTA restored high resazurin reduction rates in untreated spheroids. In conclusion, the RR assay is unsuited to quantitatively measure cellular health/cell number in compact spheroids. However, it can be used to distinguish between cytotoxic versus cytostatic compounds in spheroids. Restoration of the correlation of cell viability/number to resazurin reduction capacity can be achieved by disruption of tight junctions.

Characterization of SVEP1, KIAA, and SRPX2 in an in vitro cell culture model of endotoxemia.

To assess the influence of unknown factors in endotoxemia, a conditioned medium, achieved by the stimulation of THP1 monocytes with lipopolysaccharide (LPS) [4h], was used for the stimulation of human umbilical vein endothelial cells (HUVECs) [16h]. SVEP1, KIAA0247, and SRPX2 were selected after microarray analysis. To study their possible functions, siRNAs of SVEP1, KIAA0247, or SRPX2 were used for the transfection of HUVECs and cells were stimulated with conditioned medium [16h]. Inhibition of SVEP1 expression resulted in an increase of soluble intercellular adhesion molecule (sICAM) 1 (10%) and soluble E-selectin (sE-selectin) (19%). Inhibition of SRPX2 led to an increase of sICAM (11%) and sE-selectin (14%). KIAA0247 negative HUVECs showed a decrease in monocyte chemoattractant protein (MCP) 1 of 16%. SVEP1 and SRPX2 seemed to act as regulators of ICAM1 and E-selectin shedding and influence the expression of membrane bound adhesion molecules.