QHREDGS Enhances Tube Formation, Metabolism and Survival of Endothelial Cells in Collagen-Chitosan Hydrogels

Cell survival in complex, vascularized tissues, has been implicated as a major bottleneck in advancement of therapies based on cardiac tissue engineering. This limitation motivates the search for small, inexpensive molecules that would simultaneously be cardio-protective and vasculogenic. Here, we present peptide sequence QHREDGS, based upon the fibrinogen-like domain of angiopoietin-1, as a prime candidate molecule. We demonstrated previously that QHREDGS improved cardiomyocyte metabolism and mitigated serum starved apoptosis. In this paper we further demonstrate the potency of QHREDGS in its ability to enhance endothelial cell survival, metabolism and tube formation. When endothelial cells were exposed to the soluble form of QHREDGS, improvements in endothelial cell barrier functionality, nitric oxide production and cell metabolism (ATP levels) in serum starved conditions were found. The functionality of the peptide was then examined when conjugated to collagen-chitosan hydrogel, a potential carrier for in vivo application. The presence of the peptide in the hydrogel mitigated paclitaxel induced apoptosis of endothelial cells in a dose dependent manner. Furthermore, the peptide modified hydrogels stimulated tube-like structure formation of encapsulated endothelial cells. When integrin αvβ3 or α5β1were antibody blocked during cell encapsulation in peptide modified hydrogels, tube formation was abolished. Therefore, the dual protective nature of the novel peptide QHREDGS may position this peptide as an appealing augmentation for collagen-chitosan hydrogels that could be used for biomaterial delivered cell therapies in the settings of myocardial infarction

Vascular Progenitor Cells Isolated from Human Embryonic Stem Cells

Invariant left-right (LR) patterning or chirality is critical for embryonic development. The loss or reversal of LR asymmetry is often associated with malformations and disease. Although several theories have been proposed, the exact mechanism of the initiation of the LR symmetry has not yet been fully elucidated. Recently, chirality has been detected within single cells as well as multicellular structures using several in vitro approaches. These studies demonstrated the universality of cell chirality, its dependence on cell phenotype, and the role of physical boundaries. In this review, we discuss the theories for developmental LR asymmetry, compare various in vitro cell chirality model systems, and highlight possible roles of cell chirality in stem cell differentiation. We emphasize that the in vitro cell chirality systems have great promise for helping unveil the nature of chiral morphogenesis in development

Linear QHREDGS Peptide Decreases Endothelial Cell Permeability.

<p>(A) HMVEC-D were cultured on collagen I and permeability was assessed using a Transwell assay (N = 3/group). Endothelial cells were serum starved (1 hr) and FITC labeled albumin was added to the top well and cells were incubated. QHREDGS or scrambled (DQSHER) peptides at 400 µM or control (PBS) were added (1 hr). Thrombin was added to induce permeability and the amount of FITC-linked albumin in the bottom well was quantified (Pa/hr) (3 hr). Peptide QHREDGS reduced HMVEC-D permeability (*P = 0.004) compared to control and scrambled peptide (**P = 0.001) groups. (B) Transendothelial electrical resistance was measured in HMVEC-D cultured on collagen I. HMVEC-D were serum starved and QHREDGS or scrambled (DQSHER) peptides at 400 µM or control (PBS) were added (1 hr). Thrombin was added to reduce resistance and resistance was measured as a function of time (N = 2/group). Peptide QHREDGS increased QHREDGS resistance compared to control and scrambled peptides. Red coloured triangles indicate normalized resistance values for the QHREDGS treated group.</p

Linear QHREDGS Peptide Increases Endothelial Cell Survival and Energetics.

<p>(A-B) MS1 endothelial cells were incubated with 500 µM QHREDGS or scrambled QHREDGS peptide (DQSHER) or control (PBS) overnight. (A) Cells were incubated for 4 hr with MTT, absorbances measured at 570 nm, and values normalized to cell number (N = 5/group). Peptide QHREDGS increased MS1 endothelial cell viability (*P = 0.001) as indicated by the increased processing of MTT by mitochondria. (B) QHREDGS peptide increased endothelial cell ATP levels, whereas scrambled QHREDGS had no such effect (*P = 0.03). (C) MS1 endothelial cells were incubated with 500 µM QHREDGS or scrambled QHREDGS peptide or control (PBS) for 3 hr. QHREDGS peptide increased endothelial cell NADH/NADPH levels (*P = 0.05) suggesting increased cellular energetics. Optical density (OD).</p

QHREDGS Modified Collagen-Chitosan Hydrogels Support Expression of CD31 and VE-CAD in Endothelial Cells Characterization of HUVECs on collagen-chitosan hydrogels with different peptide concentrations after 4 days of cultivation.

<p>187,500 cells were seeded on the freshly made hydrogels. (A) HUVECs stained for CD31 (green) and cell nucleus (blue) in control, 100 µM peptide and 650 µM peptide hydrogels. 20X magnification with scale bar of 50 µm and higher magnification inset scale bar of 10 µm. (B) HUVECs stained for VE-CAD (red) and cell nucleus (blue) stained in control, 100 µM peptide, and 650 µM peptide hydrogels. 40X magnification with scale bar of 20 µm. (D) Average cell area. (E) Average cell number per cm².</p

QHREDGS Modified Collagen-Chitosan Hydrogels Enhance Tube-Like Structure Formation of Encapsulated HUVECs.

<p>Encapsulation of HUVECs within the control hydrogel and two peptide modified hydrogel groups. (A) HUVECs stained with CFDA-SE for live cells (green). Images taken every other day to show the progression of HUVEC constructs forming within the hydrogel. (B) Average longest construct path length determined on day 4. (C) Average total path length of constructs determined on day 4. * P<0.05. All images are taken at 4X magnification. Scale bar is 200 µm.</p

QHREDGS Provides a Protective Effect on Endothelial Cells Undergoing Apoptosis on Collagen-Chitosan Hydrogels.

<p>Caspase-3/7 activity, normalized to cell number, is shown for each of the three hydrogel-peptide concentrations. After three days of HUVEC cultivation, each hydrogel group was treated three different ways for 15 hr. The treatments were: PBS control, DMSO carrier and Taxol 2.5 µM. * P<0.05, # P = 0.051.</p

QHREDGS Modified Collagen-Chitosan Hydrogels Expedite Kinetics of Endothelial Cell Tube Formation.

<p>Encapsulation of HUVECs within the control, 100 µM peptide and 650 µM peptide hydrogel groups with HUVEC membranes stained with CellTracker dye (red). Images taken at 6, 18, and 24 hr to show the progression of HUVEC constructs forming within the hydrogel. All images were taken at 20X magnification. Scale bar is 50 µm.</p

Blocking αvβ3 or α5β1 Integrin Receptor Inhibits Formation of QHREDGS Tube-Like Structures by Encapsulated HUVECs.

<p>Encapsulation of HUVECs within the control hydrogel and two peptide modified hydrogel groups for two days in the presence of either an Igg control, αvβ3 or α5β1 antibody. HUVECs stained with CFDA<b>-</b>SE for live cells (green). Images taken at the end of the two day cultivation to examine tube-like structure formation. (A) Images taken at 4X magnification. Scale bar is 200 µm. (B) Images taken at 10X magnification. Scale bar is 100 µm.</p

Cultivation of HUVECs on QHREDGS Modified Collagen-Chitosan Hydrogel Leads to Improved Viability.

<p>Characterization of HUVECs on collagen-chitosan hydrogels with different peptide concentrations after 4 days of cultivation. 187,500 cells were seeded on the freshly made hydrogels. (A) Control hydrogel, (B) 100 µM peptide hydrogel and (C) 650 µM peptide hydrogel with HUVECs stained for live/dead imaging with CFDA-SE for live cells (green) and PI for dead cells (red). (D) Percentage of live cells. All images are 20X magnification with scale bar of 50 µm. *P<0.05.</p