Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices

Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC

iPSC-Derived Vascular Cell Spheroids as Building Blocks for Scaffold-Free Biofabrication

Recently a protocol is established to obtain large quantities of human induced pluripotent stem cells (iPSC)-derived endothelial progenitors, called endothelial colony forming cells (ECFC), and of candidate smooth-muscle forming cells (SMFC). Here, the suitability for assembling in spheroids, and in larger 3D cell constructs is tested. iPSC-derived ECFC and SMFC are labeled with tdTomato and eGFP, respectively. Spheroids are formed in ultra-low adhesive wells, and their dynamic proprieties are studied by time-lapse microscopy, or by confocal microscopy. Spheroids are also tested for fusion ability either in the wells, or assembled on the Regenova 3D bioprinter which laces them in stainless steel micro-needles (the “Kenzan” method). It is found that both ECFC and SMFC formed spheroids in about 24 h. Fluorescence monitoring indicated a continuous compaction of ECFC spheroids, but stabilization in those prepared from SMFC. In mixed spheroids, the cell distribution changed continuously, with ECFC relocating to the core, and showing pre-vascular organization. All spheroids have the ability of in-well fusion, but only those containing SMFC are robust enough to sustain assembling in tubular structures. In these constructs a layered distribution of alpha smooth muscle actin-positive cells and extracellular matrix deposition is found. In conclusion, iPSC-derived vascular cell spheroids represent a promising new cellular material for scaffold-free biofabrication

Proteomic Analysis Reveals GLUT1 to be a Novel Discriminating Marker of Human Arterial Endothelium In vivo and loss of Venous Identity in Cell Culture

Despite greatly improved understanding of endothelial heterogeneity, the number of molecules discriminating human arterial and venous endothelium remains limited. Indeed, there have been few reports validating markers proposed in animal model studies in freshly isolated human tissues. We report here the global characterization of freshly isolated human umbilical arterial and venous endothelial cell (HUAECs and HUVECs) plasma membrane proteins using an experimentally validated label-free quantitative LC-MS/MS platform. ECs were harvested by enzymatic digestion and purified by flow cytometry (CD31+, CD45-) prior to quantitative analyses. Following plasma membrane fractionation, we identified 4,300 proteins with high confidence using LC-MS/MS. GLUT1, an important regulator of endothelial function, was found to be up regulated in HUAECs 2.6 fold at the protein level and confirmed at the mRNA level using qRT-PCR. Using tissue immunohistochemistry, we discovered that GLUT1 expression was restricted to the cell surface in human arterial endothelium using umbilical cord and adult peripheral vascular sections. Importantly, GLUT1 mRNA levels decreased 20 fold in cultured arterial ECs within 48hrs of culture and continued to decline for 12 days in vitro. Principal Component Analyses demonstrated a profound effect of cell culture on protein expression signatures with cultured HUVECs, fresh HUVECS, and fresh HUAECs equally distinct. GLUT1 expression serves as a robust discriminator of arterial versus venous ECs in vivo and marks a loss of venous EC identity in vitro.</p

Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells.

Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly

ExoY activity causes a decrease in microtubules in PMVECs.

<p>[<b>A</b>] PMVECs infected with <i>P.aeruginosa</i> expressing either non-functional K81M mutant ExoY (ExoY^K81M; center) or wild type ExoY (right) were observed following processing for anti-tubulin immunofluorescence microscopy. Uninfected cells are also shown (left). Bar = 10 µm. [<b>B</b>] Levels of polymerized tubulin (P) and unpolymerized soluble tubulin (S) were quantified by immunoblot analysis using antibody against α-tubulin. Extracts obtained from untreated cells (Ctr) and from PMVECs infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺ are shown. The ratio of polymerized tubulin to soluble tubulin was significantly less in cells containing wild type ExoY⁺ [0.29±0.06 <i>vs.</i> 0.44±0.05 (ExoY^K81M) and 0.49±0.08 (Ctr); n = 5; P<0.05 compared to both ExoY^K81M and untreated control].</p

ExoY activity does not noticeably affect microtubule assembly from PMVEC centrosomes.

<p>PMVECs were infected with <i>P. aeruginosa</i> expressing either ExoY^K81M (middle panel) or ExoY⁺ (left panel). Untreated cells (Ctr) and infected cells were extracted to remove soluble proteins and then purified rat brain tubulin was added and microtubule nucleation was initiated by incubating at 37°C for 15 min. The preparations then were fixed and labeled with antitubulin antibodies. Centrosome nucleation of microtubules is shown by arrows. Bar = 10 µm.</p

ExoY activity affects microtubule assembly in PMVECs.

<p>PMVECs were infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺. Untreated control cells (Ctr) and infected cells were then placed on ice to induce microtubule disassembly. Microtubule re-growth was initiated by transferring the cells to 37°C. [<b>A.</b>] Individual coverslips were fixed either at the time of transfer (T = 0) or at varying times after transfer to 37°C. The coverslips then were labeled with antitubulin antibodies. Cells at 4 and 8 minutes post-transfer are shown; microtubule growth is apparent by 4 minutes and peripheral microtubules are resolved by 8 minutes in untreated and K81M infected cells. Microtubule re-growth lagged significantly in cells intoxicated with wt ExoY. Bar = 10 µm. [<b>B.</b>] Polymerized (P) and soluble unpolymerized (S) tubulin levels were quantified by immunoblot analysis using antitubulin antibodies. To obtain soluble and polymer fractions, control cells and cells infected with bacteria expressing either ExoY^K81M or ExoY⁺ were extracted at 8 minutes post-transfer to 37°C. The ratio of polymerized tubulin to soluble tubulin was significantly less in cells containing wild type ExoY [0.10±0.06 <i>vs.</i> 0.36±0.10 (K81M) and 0.41±0.09 (Ctr); n = 4; P<0.05 compared to both K81M and untreated cells]. [<b>C.</b>] Tau levels are unchanged following cold treatment to disassemble microtubules. Cells were treated with cold and then whole extracts were collected from control cells (Ctr) and from cells that were intoxicated with either ExoY⁺ or ExoY^K81M. The extracts were then probed for tau levels using polyclonal anti-tau antibody.</p