Peptide interfacial biomaterials improve endothelial cell adhesion and spreading on synthetic polyglycolic acid materials.

Resorbable scaffolds such as polyglycolic acid (PGA) are employed in a number of clinical and tissue engineering applications owing to their desirable property of allowing remodeling to form native tissue over time. However, native PGA does not promote endothelial cell adhesion. Here we describe a novel treatment with hetero-bifunctional peptide linkers, termed "interfacial biomaterials" (IFBMs), which are used to alter the surface of PGA to provide appropriate biological cues. IFBMs couple an affinity peptide for the material with a biologically active peptide that promotes desired cellular responses. One such PGA affinity peptide was coupled to the integrin binding domain, Arg-Gly-Asp (RGD), to build a chemically synthesized bimodular 27 amino acid peptide that mediated interactions between PGA and integrin receptors on endothelial cells. Quartz crystal microbalance with dissipation monitoring (QCMD) was used to determine the association constant (K (A) 1 x 10(7) M(-1)) and surface thickness (~3.5 nm). Cell binding studies indicated that IFBM efficiently mediated adhesion, spreading, and cytoskeletal organization of endothelial cells on PGA in an integrin-dependent manner. We show that the IFBM peptide promotes a 200% increase in endothelial cell binding to PGA as well as 70-120% increase in cell spreading from 30 to 60 minutes after plating.

NgBR is essential for endothelial cell glycosylation and vascular development.

NgBR is a transmembrane protein identified as a Nogo-B-interacting protein and recently has been shown to be a subunit required for cis-prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial-specific NgBR knockout embryos. Here, we show that endothelial-specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE-cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo-B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.

Porcine endothelial cells cocultured with smooth muscle cells became procoagulant in vitro.

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

A PK2/Bv8/PROK2 antagonist suppresses tumorigenic processes by inhibiting angiogenesis in glioma and blocking myeloid cell infiltration in pancreatic cancer.

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy.

Tissue engraftment of hypoxic-preconditioned adipose-derived stem cells improves flap viability.

Adipose-derived stem cells (ASCs) have the ability to release multiple growth factors in response to hypoxia. In this study, we investigated the potential of ASCs to prevent tissue ischemia. We found conditioned media from hypoxic ASCs had increased levels of vascular endothelial growth factor (VEGF) and enhanced endothelial cell tubule formation. To investigate the effect of injecting rat ASCs into ischemic flaps, 21 Lewis rats were divided into three groups: control, normal oxygen ASCs (10(6) cells), and hypoxic preconditioned ASCs (10(6) cells). At the time of flap elevation, the distal third of the flap was injected with the treatment group. At 7 days post flap elevation, flap viability was significantly improved with injection of hypoxic preconditioned ASCs. Cluster of differentiation-31-positive cells were more abundant along the margins of flaps injected with ASCs. Fluorescent labeled ASCs localized aside blood vessels or throughout the tissue, dependent on oxygen preconditioning status. Next, we evaluated the effect of hypoxic preconditioning on ASC migration and chemotaxis. Hypoxia did not affect ASC migration on scratch assay or chemotaxis to collagen and laminin. Thus, hypoxic preconditioning of injected ASCs improves flap viability likely through the effects of VEGF release. These effects are modest and represent the limitations of cellular and growth factor-induced angiogenesis in the acute setting of ischemia.

Interstitial engraftment of adipose-derived stem cells into an acellular dermal matrix results in improved inward angiogenesis and tissue incorporation.

Acellular dermal matrices (ADM) are commonly used in reconstructive procedures and rely on host cell invasion to become incorporated into host tissues. We investigated different approaches to adipose-derived stem cells (ASCs) engraftment into ADM to enhance this process. Lewis rat adipose-derived stem cells were isolated and grafted (3.0 × 10(5) cells) to porcine ADM disks (1.5 mm thick × 6 mm diameter) using either passive onlay or interstitial injection seeding techniques. Following incubation, seeding efficiency and seeded cell viability were measured in vitro. In addition, Eighteen Lewis rats underwent subcutaneous placement of ADM disk either as control or seeded with PKH67 labeled ASCs. ADM disks were seeded with ASCs using either onlay or injection techniques. On day 7 and or 14, ADM disks were harvested and analyzed for host cell infiltration. Onlay and injection techniques resulted in unique seeding patterns; however cell seeding efficiency and cell viability were similar. In-vivo studies showed significantly increased host cell infiltration towards the ASCs foci following injection seeding in comparison to control group (p < 0.05). Moreover, regional endothelial cell invasion was significantly greater in ASCs injected grafts in comparison to onlay seeding (p < 0.05). ADM can successfully be engrafted with ASCs. Interstitial engraftment of ASCs into ADM via injection enhances regional infiltration of host cells and angiogenesis, whereas onlay seeding showed relatively broad and superficial cell infiltration. These findings may be applied to improve the incorporation of avascular engineered constructs.

A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells.

Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for angiogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular endothelial growth factor (VEGF)-A or changes in TGF-β1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45+ cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements, however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression, as evidenced by a lack of significant differences in total segment length (p=0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45+ cells and improves upon the addition of soluble growth factors.

Human uterine leiomyoma-derived fibroblasts stimulate uterine leiomyoma cell proliferation and collagen type I production, and activate RTKs and TGF beta receptor signaling in coculture.

BACKGROUND: Uterine leiomyomas (fibroids) are benign smooth muscle tumors that often contain an excessive extracellular matrix (ECM). In the present study, we investigated the interactions between human uterine leiomyoma (UtLM) cells and uterine leiomyoma-derived fibroblasts (FB), and their importance in cell growth and ECM protein production using a coculture system. RESULTS: We found enhanced cell proliferation, and elevated levels of ECM collagen type I and insulin-like growth factor-binding protein-3 after coculturing. There was also increased secretion of vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor-2, and platelet derived growth factor A and B in the media of UtLM cells cocultured with FB. Protein arrays revealed increased phosphorylated receptor tyrosine kinases (RTKs) of the above growth factor ligands, and immunoblots showed elevated levels of the RTK downstream effector, phospho-mitogen activated protein kinase 44/42 in cocultured UtLM cells. There was also increased secretion of transforming growth factor-beta 1 and 3, and immunoprecipitated transforming growth factor-beta receptor I from cocultured UtLM cells showed elevated phosphoserine expression. The downstream effectors phospho-small mothers against decapentaplegic -2 and -3 protein (SMAD) levels were also increased in cocultured UtLM cells. However, none of the above effects were seen in normal myometrial cells cocultured with FB. The soluble factors released by tumor-derived fibroblasts and/or UtLM cells, and activation of the growth factor receptors and their pathways stimulated the proliferation of UtLM cells and enhanced the production of ECM proteins. CONCLUSIONS: These data support the importance of interactions between fibroid tumor cells and ECM fibroblasts in vivo, and the role of growth factors, and ECM proteins in the pathogenesis of uterine fibroids.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

An active learning approach for rapid characterization of endothelial cells in human tumors.

Currently, no available pathological or molecular measures of tumor angiogenesis predict response to antiangiogenic therapies used in clinical practice. Recognizing that tumor endothelial cells (EC) and EC activation and survival signaling are the direct targets of these therapies, we sought to develop an automated platform for quantifying activity of critical signaling pathways and other biological events in EC of patient tumors by histopathology. Computer image analysis of EC in highly heterogeneous human tumors by a statistical classifier trained using examples selected by human experts performed poorly due to subjectivity and selection bias. We hypothesized that the analysis can be optimized by a more active process to aid experts in identifying informative training examples. To test this hypothesis, we incorporated a novel active learning (AL) algorithm into FARSIGHT image analysis software that aids the expert by seeking out informative examples for the operator to label. The resulting FARSIGHT-AL system identified EC with specificity and sensitivity consistently greater than 0.9 and outperformed traditional supervised classification algorithms. The system modeled individual operator preferences and generated reproducible results. Using the results of EC classification, we also quantified proliferation (Ki67) and activity in important signal transduction pathways (MAP kinase, STAT3) in immunostained human clear cell renal cell carcinoma and other tumors. FARSIGHT-AL enables characterization of EC in conventionally preserved human tumors in a more automated process suitable for testing and validating in clinical trials. The results of our study support a unique opportunity for quantifying angiogenesis in a manner that can now be tested for its ability to identify novel predictive and response biomarkers.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.