Enhanced Endothelial Cell Research Articles

Construction of Mussel-Inspired Dopamine-Zn2+ Coating on Titanium Oxide Nanotubes to Improve Hemocompatibility, Cytocompatibility, and Antibacterial Activity.

Zinc ions (Zn2+) are a highly potent bioactive factor with a broad spectrum of physiological functions. In situ continuous and controllable release of Zn2+ from the biomaterials can effectively improve the biocompatibility and antibacterial activity. In the present study, inspired by the adhesion and protein cross-linking in the mussel byssus, with the aim of improving the biocompatibility of titanium, a cost-effective one-step metal–catecholamine assembly strategy was developed to prepare a biomimetic dopamine–Zn2+ (DA-Zn2+) coating by immersing the titanium oxide nanotube (TNT) arrays on the titanium surface prepared by anodic oxidation into an aqueous solution containing dopamine (DA) and zinc ions (Zn2+). The DA-Zn2+ coatings with the different zinc contents exhibited excellent hydrophilicity. Due to the continuous release of zinc ions from the DA-Zn2+ coating, the coated titanium oxide nanotubes displayed excellent hemocompatibility characterized by platelet adhesion and activation and hemolysis assay. Moreover, the DA-Zn2+-coated samples exhibited an excellent ability to enhance endothelial cell (EC) adhesion and proliferation. In addition, the DA-Zn2+ coating can also enhance the antibacterial activity of the nanotubes. Therefore, long-term in situ Zn2+-releasing coating of the present study could serve as the bio-surfaces for long-term prevention of thrombosis, improvement of cytocompatibility to endothelial cells, and antibacterial activity. Due to the easy operation and strong binding ability of the polydopamine on various complicated shapes, the method of the present study can be further applied to other blood contact biomaterials or implantable medical devices to improve the biocompatibility.

Tri-Layered Vascular Grafts Guide Vascular Cells' Native-like Arrangement.

Bionic grafts hold great promise for directing tissue regeneration. In vascular tissue engineering, although a large number of synthetic grafts have been constructed, these substitutes only partially recapitulated the tri-layered structure of native arteries. Synthetic polymers such as poly(l-lactide-co-ε-caprolactone) (PLCL) possess good biocompatibility, controllable degradation, remarkable processability, and sufficient mechanical strength. These properties of PLCL show great promise for fabricating synthetic vascular substitutes. Here, tri-layered PLCL vascular grafts (TVGs) composed of a smooth inner layer, circumferentially aligned fibrous middle layer, and randomly distributed fibrous outer layer were prepared by sequentially using ink printing, wet spinning, and electrospinning techniques. TVGs possessed kink resistance and sufficient mechanical properties (tensile strength, elastic modulus, suture retention strength, and burst pressure) equivalent to the gold standard conduits of clinical application, i.e., human saphenous veins and human internal mammary arteries. The stratified structure of TVGs exhibited a visible guiding effect on specific vascular cells including enhancing endothelial cell (EC) monolayer formation, favoring vascular smooth muscle cells’ (VSMCs) arrangement and elongation, and facilitating fibroblasts’ proliferation and junction establishment. Our research provides a new avenue for designing synthetic vascular grafts with polymers.

High-Efficient Vacuum Ultraviolet-Ozone Assist-Deposited Polydopamine for Poly(lactic-co-glycolic acid)-Coated Pure Zn toward Biodegradable Cardiovascular Stent Applications.

Zinc is a prospective metal for biodegradable cardiovascular stent applications, but the excessively released Zn2+ during degradation remains a huge challenge in biocompatibility. Considerable efforts have been made to develop a high-efficient surface modification method, while maintaining adhesion strength, mechanical support, and vascular compatibility. Biomimetic polydopamine (PDA) can adhere to Zn tightly, subsequently achieving robust chemical bonds with poly(lactic-co-glycolic acid) (PLGA) coating. However, the deposition of PDA on Zn depends on the controlled conditions such as a sensitive pH and a long period of time. Herein, we introduce vacuum ultraviolet-ozone (VUV/O3) assist-deposition technology to accelerate the polymerization of PDA on pure Zn, which shortens the process to 40 min at a moderate pH of 8.5 and improves the deposition rate by 1-2 orders of magnitude under sufficient active oxygen species (ROS). Additionally, PLGA/PDA coating enhances the corrosion resistance, and their effective protection maintains the mechanical properties after long-term corrosion. Moreover, the controlled Zn2+ release contributes to the superior in vitro biocompatibility, which inhibits the hemolysis rate and smooth muscle cell (SMC) proliferation. The enhanced endothelial cell (EC) proliferation is promising to promote the re-endothelialization, avoiding in-stent restenosis and neointimal hyperplasia. Such modified Zn might be a viable candidate for the treatment of cardiovascular diseases.

Substrate stiffening promotes VEGF-A functions via the PI3K/Akt/mTOR pathway

While it is now well-established that substrate stiffness regulates vascular endothelial growth factor-A (VEGF-A) mediated signaling and functions, causal mechanisms remain poorly understood. Here, we report an underlying role for the PI3K/Akt/mTOR signaling pathway. This pathway is activated on stiffer substrates, is amplified by VEGF-A stimulation, and correlates with enhanced endothelial cell (EC) proliferation, contraction, pro-angiogenic secretion, and capillary-like tube formation. In the settings of advanced age-related macular degeneration, characterized by EC and retinal pigment epithelial (RPE)-mediated angiogenesis, these data implicate substrate stiffness as a novel causative mechanism and Akt/mTOR inhibition as a novel therapeutic pathway.

SAT-LB97 MiRNA-99a and mTOR2 Mediate Enhanced Endothelial Mineralocorticoid Receptor Signaling-Induced Activation of Sodium Channel and Endothelium Stiffness

In diet induced obesity enhanced endothelial cell (EC) mineralocorticoid receptor (MR) (ECMR) and downstream sodium channel (EnNaC) activity increases oxidative stress and inflammation, thereby promoting vascular stiffness and associated impaired endothelial mediated relaxation. For example, consumption of a Western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks elevated plasma aldosterone levels and increased vascular MR expression in conjunction with increased endothelial and vascular stiffness in female mice. EC specific deletion of either the ECMR or EnNaC significantly attenuated this diet induced endothelial/vascular stiffness. Emerging information suggests that abnormal expression of miR-99a may be involved in these processes. To this point, we recently observed that aldosterone (10-7 mol/L) causes a reduction in miR-99a that was prevented by the MR antagonist, spironolactone (10µM) in in vitro ECs. By using RNA sequencing, we also demonstrated that ECMR activation reduced arterial miR-99a expression in diet induced obesity. Since the mammalian target of rapamycin (mTOR2)/SGK1 signaling pathway is involved in aldosterone activation of ENaC we then explored the effects of miR-99a on mTOR2 expression. Indeed, miR-99a reduced mTOR2. We further observed that inhibition of mTOR2 with PP242 inhibited EnNaC activity as determined by patch clamping of ECs. Collectively these data suggest that consumption of a WD induced ECMR activation and increased EnNaC activity and endothelial stiffness, in part, by reducing the tonic inhibitory effects exerted by miR-99a on mTOR2 mediated EnNaC activation.

Hypoxic Preconditioning Enhances Survival and Proangiogenic Capacity of Human First Trimester Chorionic Villus-Derived Mesenchymal Stem Cells for Fetal Tissue Engineering.

Prenatal stem cell-based regenerative therapies have progressed substantially and have been demonstrated as effective treatment options for fetal diseases that were previously deemed untreatable. Due to immunoregulatory properties, self-renewal capacity, and multilineage potential, autologous human placental chorionic villus-derived mesenchymal stromal cells (CV-MSCs) are an attractive cell source for fetal regenerative therapies. However, as a general issue for MSC transplantation, the poor survival and engraftment is a major challenge of the application of MSCs. Particularly for the fetal transplantation of CV-MSCs in the naturally hypoxic fetal environment, improving the survival and engraftment of CV-MSCs is critically important. Hypoxic preconditioning (HP) is an effective priming approach to protect stem cells from ischemic damage. In this study, we developed an optimal HP protocol to enhance the survival and proangiogenic capacity of CV-MSCs for improving clinical outcomes in fetal applications. Total cell number, DNA quantification, nuclear area test, and cell viability test showed HP significantly protected CV-MSCs from ischemic damage. Flow cytometry analysis confirmed HP did not alter the immunophenotype of CV-MSCs. Caspase-3, MTS, and Western blot analysis showed HP significantly reduced the apoptosis of CV-MSCs under ischemic stimulus via the activation of the AKT signaling pathway that was related to cell survival. ELISA results showed HP significantly enhanced the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by CV-MSCs under an ischemic stimulus. We also found that the environmental nutrition level was critical for the release of brain-derived neurotrophic factor (BDNF). The angiogenesis assay results showed HP-primed CV-MSCs could significantly enhance endothelial cell (EC) proliferation, migration, and tube formation. Consequently, HP is a promising strategy to increase the tolerance of CV-MSCs to ischemia and improve their therapeutic efficacy in fetal clinical applications.

Mussel-inspired dopamine-CuII coatings for sustained in situ generation of nitric oxide for prevention of stent thrombosis and restenosis

Nitric oxide (NO) is a highly potent, yet short-lived bioactive molecule with a broad spectrum of physiological functions. Continuous and controllable in situ generation of NO from vascular stent surface can effectively prevent restenosis and thrombosis after its implantation. In this study, inspired by the adhesion and protein cross-linking in the mussel byssus, through immersing the stents into an aqueous solution with dopamine (DA) and copper ions (CuII), we developed a one-step metal-catecholamine assembled strategy to prepare a durable in situ NO-generating biomimetic coating (DA-CuII). Due to the high NO catalytic efficacy and robust chelation of CuII into the DA-CuII network, the coated stents exhibited excellent hemocompatibility. The coating also catalytically decomposed endogenous S-nitrosothiols (RSNOs) from fresh blood, and locally generated NO for over 30 days with a flux comparable to its physiological range (0.5–4 × 10−10 mol × cm−2 × min−1). Moreover, the optimized biomimetic coatings displayed specific cell selectivity to significantly enhance endothelial cell (EC) growth while substantially inhibit smooth muscle cell (SMC) growth and migration. This feature impressively promoted regeneration of a new endothelial cell layer after stent implantation, hence improved the anti-thrombogenic and anti-restenosis qualities of vascular stents in vivo. We envision that our long-term in situ NO-generating coatings could serve as biosurfaces for long-term prevention of stent thrombosis and restenosis.

CysLT2R is a novel therapeutic target for tumor angiogenesis, growth and metastasis

Angiogenesis plays a vital role in numerous diseases including cancer, atherosclerosis, diabetic retinopathy and age‐related macular degeneration. Although chronic inflammation mediated by immune cells has been known to play a role in neoplastic transformation, there exists limited information on how inflammatory mediators can cause endothelial dysfunction and their role in promoting angiogenesis. Cysteinyl leukotrienes (Cys‐LTs: LTC4, LTD4 and LTE4) are pro‐inflammatory mediators derived from arachidonic acid and signal through two well characterized receptors, CysLT1R and CysLT2R. Cys‐LTs have been shown to be involved in several human cancers including breast and melanoma. However, the receptors involved and the mechanism of action still remains elusive. We have recently shown that LTD4 can enhance endothelial cell (EC) contraction and permeability via CysLT2R in a Rho‐dependent pathway. Here, we present another seminal observation that LTD4 can enhance angiogenesis in vitro and in vivo via CysLT2R. Since enhanced angiogenesis is required for solid tumor growth, we investigated the role of CysLTR in tumor angiogenesis, tumor progression and metastasis using mice lacking CysLT1R (CysLT1RKO) and CysLT2R (CysLT2RKO). We observed enhanced CysLT2R expression but not CysLT1R in wild type (WT) tumors, in line with the involvement of CysLT2R in endothelial dysfunction. Further, tumor growth and angiogenesis were significantly decreased in CysLT2RKO mice compared to WT and CysLT1RKO mice in subcutaneous Lewis lung carcinoma (LLC) tumor model. Furthermore, though few in number, tumor vessels in CysLT2RKO mice exhibited intact pericyte coverage and reduced permeability with simultaneous reduction in tumor cell metastasis to the lung. Finally, blocking CysLT2R using a specific CysLT2R antagonist BaycysLT2 recapitulated the results obtained with CysLT2RKO mice. Our results suggest that CysLT2R promotes tumor angiogenesis and growth as well as metastasis to the lung by disrupting vascular integrity and targeting CysLT2R could offer novel VEGF‐independent therapeutic strategy for the treatment of cancer and other angiogenic disorders.Support or Funding InformationJames Foght Professorship Support, AHA GIA 15GRNT 25670004This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration.

We investigated the role of exosomes derived from maternal and umbilical cord blood in the regulation of angiogenesis. We report here that both maternal exosomes (MEs) and umbilical exosomes (UEs) significantly enhance HUVEC proliferation, migration, and tube formation. Importantly, ME-treated HUVECs (MEXs) displayed significantly increased migration, but not proliferation or tube formation, compared with UE-treated HUVECs (UEXs). We found that the expression of a subset of migration-related microRNAs (miRNAs), including miR-210-3p, miR-376c-3p, miR-151a-5p, miR-296-5p, miR-122-5p, and miR-550a-5p, among others, were significantly increased or decreased in UEs, and this altered expression was likely correlated with the differential regulation of HUVEC migration. We also found that the mRNA expression of hepatocyte growth factor (HGF) was up-regulated in MEXs and UEXs and, moreover, that inhibiting HGF partially abolished the enhanced cell migration induced by UEs. Our results suggest that both MEs and UEs greatly enhanced endothelial cell (EC) functions and differentially regulated EC migration, which was mostly attributed to the different expression profiles of exosomal miRNA. These findings highlight the importance of exosomes in the regulation of angiogenesis during pregnancy. Exosomal miRNAs, in particular, may be of great significance for the regulation of angiogenesis in maintaining normal pregnancy.-Jia, L., Zhou, X., Huang, X., Xu, X., Jia, Y., Wu, Y., Yao, J., Wu, Y., Wang, K. Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration.

Loss of osteoglycin promotes angiogenesis in limb ischaemia mouse models via modulation of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 signalling pathway.

Osteoglycin (OGN) has been noted for its implication in cardiovascular disease in recent studies. However, the relationship between OGN and angiogenesis remains unknown. Therefore, we aimed to investigate the effect of OGN on ischaemia-induced angiogenesis and to address the underlying mechanisms. The expression of OGN was decreased in a limb ischaemia mouse model. OGN knockout (KO) mice were used to further understand the role of OGN after ischaemia. The perfusion recovery rate after femoral artery ligation was higher in OGN KO mice than in wild-type (WT) mice. The capillary density in the gastrocnemius muscle of the ischaemic limb was also higher in OGN KO mice. Moreover, ex vivo aortic ring explants from OGN KO mice exhibited stronger angiogenic sprouting than those from WT mice. In human umbilical vein endothelial cells (HUVECs), OGN knockdown enhanced endothelial cell (EC) activation, including tube formation, proliferation, and migration. In contrast, OGN overexpression inhibited HUVEC activation. Mechanistic studies revealed that OGN associates with vascular endothelial growth factor receptor 2 (VEGFR2) and negatively regulates the interaction of vascular endothelial growth factor (VEGF) and VEGFR2, thereby negatively modulating the activation of VEGFR2 and its downstream signalling pathways. Consistently, the pro-angiogenic effect of OGN KO was abrogated by VEGFR2 inhibition, supporting the critical role of VEGFR2 signalling in OGN-mediated regulation of angiogenic function. OGN plays a critical role in negatively regulating ischaemia-induced angiogenesis by inhibiting VEGF-VEGFR2 signalling and thereby attenuating EC tube formation, proliferation, and migration. Thus, OGN may be a novel therapeutic target for ischaemic vascular diseases.

CTHRC1 promotes angiogenesis by recruiting Tie2-expressing monocytes to pancreatic tumors.

CTHRC1 (collagen triple-helix repeat-containing 1), a protein secreted during the tissue-repair process, is highly expressed in several malignant tumors, including pancreatic cancer. We recently showed that CTHRC1 has an important role in the progression and metastasis of pancreatic cancer. Although CTHRC1 secretion affects tumor cells, how it promotes tumorigenesis in the context of the microenvironment is largely unknown. Here we identified a novel role of CTHRC1 as a potent endothelial activator that promotes angiogenesis by recruiting bone marrow-derived cells to the tumor microenvironment during tumorigenesis. Recombinant CTHRC1 (rCTHRC1) enhanced endothelial cell (EC) proliferation, migration and capillary-like tube formation, which was consistent with the observed increases in neovascularization in vivo. Moreover, rCTHRC1 upregulated angiopoietin-2 (Ang-2), a Tie2 receptor ligand, through ERK-dependent activation of AP-1 in ECs, resulting in recruitment of Tie2-expressing monocytes (TEMs) to CTHRC1-overexpressing tumor tissues. Treatment with a CTHRC1-neutralizing antibody-abrogated Ang-2 expression in the ECs in vitro. Moreover, administration of a CTHRC1-neutralizing antibody to a xenograft mouse model reduced the tumor burden and infiltration of TEMs in the tumor tissues, indicating that blocking the CTHRC1/Ang-2/TEM axis during angiogenesis inhibits tumorigenesis. Collectively, our findings support the hypothesis that CTHRC1 induction of the Ang-2/Tie2 axis mediates the recruitment of TEMs, which are important for tumorigenesis and can be targeted to achieve effective antitumor responses in pancreatic cancers.

Abstract 3390: CTHRC1 promotes angiogenesis by recruiting Tie2-expressing monocytes to pancreatic tumors

Abstract CTHRC1 (collagen triple helix repeat-containing 1), a protein secreted during the tissue repair process, is highly expressed in several malignant tumors, including pancreatic cancer. We recently showed that CTHRC1 plays an important role in the progression and metastasis of pancreatic cancer. Although CTHRC1 secretion affects tumor cells, how it promotes tumorigenesis in the context of the microenvironment is largely unknown. Here, we demonstrate a novel role of CTHRC1 as a potent endothelial activator, promoting angiogenesis by recruiting bone marrow-derived cells into the tumor microenvironment during tumorigenesis. Recombinant CTHRC1 (rCTHRC1) enhanced endothelial cell (EC) proliferation, migration and capillary-like tube formation, consistent with observed increases in neovascularization in vivo. Moreover, rCTHRC1 induced upregulation of angiopoietin-2 (Ang-2), a Tie2 receptor ligand, through ERK-dependent activation of AP-1 in ECs, resulting in recruitment of Tie2-expressing monocytes (TEMs) to CTHRC1-overexpressing tumor tissues. Treatment with a CTHRC1-neutralizing antibody abrogated Ang-2 expression in ECs in vitro. Moreover, CTHRC1-neutralizing antibody treatment in a xenograft mouse model reduced tumor burden and infiltration of TEMs in tumor tissues, indicating that blocking the CTHRC1/Ang-2/TEM axis during angiogenesis inhibits tumorigenesis. Collectively, our findings support the conclusion that CTHRC1 induction of the Ang-2/Tie2 axis mediates the recruitment of TEMs, which are important for tumorigenesis and can be targeted to achieve effective antitumor responses in pancreatic cancers. Citation Format: Seongyea Jo, Jaemin Lee, jinhoi song. CTHRC1 promotes angiogenesis by recruiting Tie2-expressing monocytes to pancreatic tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3390.

Concurrent and Sustained Delivery of FGF2 and FGF9 from Electrospun Poly(ester amide) Fibrous Mats for Therapeutic Angiogenesis.

Therapeutic angiogenesis has emerged as a potential strategy to treat ischemic vascular diseases. However, systemic or local administration of growth factors is usually inefficient for maintaining the effective concentration at the site of interest due to their rapid clearance or degradation. In this study, we report a differential and sustained release of an angiogenic factor, fibroblast growth factor-2 (FGF2), and an arteriogenic factor, fibroblast growth factor-9 (FGF9), from α-amino acid-derived biodegradable poly(ester amide) (PEA) fibers toward targeting neovessel formation and maturation. FGF2 and FGF9 were dual loaded using a mixed blend and emulsion electrospinning technique and exhibited differential and sustained release from PEA fibers over 28 days with preserved bioactivity. In vitro angiogenesis assays showed enhanced endothelial cell (EC) tube formation and directed migration of smooth muscle cells (SMCs) to platelet-derived growth factor (PDGF)-BB and stabilized EC/SMC tube formation. FGF2/FGF9-loaded PEA fibers did not induce inflammatory responses in vitro using human monocytes or in vivo after their subcutaneous implantation into mice. Histological examination showed that FGF2/FGF9-loaded fibers induced cell niche recruitment around the site of implantation. Furthermore, controlled in vivo delivery of FGF9 to mouse tibialis anterior (TA) muscle resulted in a dose-dependent expansion of mesenchymal progenitor-like cell layers and extracellular matrix deposition. Our data suggest that the release of FGF2 and FGF9 from PEA fibers offers an efficient differential and sustained growth factor delivery strategy with relevance to therapeutic angiogenesis.

TCGA data and patient-derived orthotopic xenografts highlight pancreatic cancer-associated angiogenesis.

Pancreatic ductal adenocarcinomas (PDACs) overexpress pro-angiogenic factors but are not viewed as vascular. Using data from The Cancer Genome Atlas we demonstrate that a subset of PDACs exhibits a strong pro-angiogenic signature that includes 37 genes, such as HDAC9, that are overexpressed in PDAC arising in KRC mice, which express mutated Kras and lack RB. Moreover, patient-derived orthotopic xenografts can exhibit tumor angiogenesis, whereas conditioned media (CM) from KRC-derived pancreatic cancer cells (PCCs) enhance endothelial cell (EC) growth and migration, and activate canonical TGF-β signaling and STAT3. Inhibition of the type I TGF-β receptor with SB505124 does not alter endothelial activation in vitro, but decreases pro-angiogenic gene expression and suppresses angiogenesis in vivo. Conversely, STAT3 silencing or JAK1-2 inhibition with ruxolitinib blocks CM-enhanced EC proliferation. STAT3 disruption also suppresses endothelial HDAC9 and blocks CM-induced HDAC9 expression, whereas HDAC9 re-expression restores CM-enhanced endothelial proliferation. Moreover, ruxolitinib blocks mitogenic EC/PCC cross-talk, and suppresses endothelial p-STAT3 and HDAC9, and PDAC progression and angiogenesis in vivo, while markedly prolonging survival of KRC mice. Thus, targeting JAK1-2 with ruxolitinib blocks a final pathway that is common to multiple pro-angiogenic factors, suppresses EC-mediated PCC proliferation, and may be useful in PDACs with a strong pro-angiogenic signature.

Endothelial cell response to chemical, biological, and physical cues in bioactive hydrogels.

The highly tunable biological, chemical, and physical properties of bioactive hydrogels enable their use in an array of tissue engineering and drug delivery applications. Systematic modulation of these properties can be used to elucidate key cell-material interactions to improve therapeutic effects. For example, the rate and extent of endothelialization are critical to the long-term success of many blood-contacting devices. To this end, we have developed a bioactive hydrogel that could be used as coating on cardiovascular devices to enhance endothelial cell (EC) adhesion and migration. The current work investigates the relative impact of hydrogel variables on key endothelialization processes. The bioactive hydrogel is based on poly(ethylene glycol) (PEG) and a streptococcal collagen-like (Scl2-2) protein that has been modified with integrin α1β1 and α2β1 binding sites. The use of PEG hydrogels allows for incorporation of specific bioactive cues and independent manipulation of scaffold properties. The selective integrin binding of Scl2-2 was compared to more traditional collagen-modified PEG hydrogels to determine the effect of integrin binding on cell behavior. Protein functionalization density, protein concentration, and substrate modulus were independently tuned with both Scl2-2 and collagen to determine the effect of each variable on EC adhesion, spreading, and migration. The findings here demonstrate that increasing substrate modulus, decreasing functionalization density, and increasing protein concentration can be utilized to increase EC adhesion and migration. Additionally, PEG-Scl2-2 hydrogels had higher migration speeds and proliferation over 1 week compared with PEG-collagen gels, demonstrating that selective integrin binding can be used to enhance cell-material interactions. Overall, these studies contribute to the understanding of the effects of matrix cues on EC interactions and demonstrate the strong potential of PEG-Scl2-2 hydrogels to promote endothelialization of blood-contacting devices.

Mussel-inspired one-step adherent coating rich in amine groups for covalent immobilization of heparin: hemocompatibility, growth behaviors of vascular cells, and tissue response.

Heparin, an important polysaccharide, has been widely used for coatings of cardiovascular devices because of its multiple biological functions including anticoagulation and inhibition of intimal hyperplasia. In this study, surface heparinization of a commonly used 316L stainless steel (SS) was explored for preparation of a multifunctional vascular stent. Dip-coating of the stents in an aqueous solution of dopamine and hexamethylendiamine (HD) (PDAM/HD) was presented as a facile method to form an adhesive coating rich in primary amine groups, which was used for covalent heparin immobilization via active ester chemistry. A heparin grafting density of about 900 ng/cm(2) was achieved with this method. The retained bioactivity of the immobilized heparin was confirmed by a remarkable prolongation of the activated partial thromboplastin time (APTT) for about 15 s, suppression of platelet adhesion, and prevention of the denaturation of adsorbed fibrinogen. The Hep-PDAM/HD also presented a favorable microenvironment for selectively enhancing endothelial cell (EC) adhesion, proliferation, migration and release of nitric oxide (NO), and at the same time inhibiting smooth muscle cell (SMC) adhesion and proliferation. Upon subcutaneous implantation, the Hep-PDAM/HD exhibited mitigated tissue response, with thinner fibrous capsule and less granulation formation compared to the control 316L SS. This number of unique functions qualifies the heparinized coating as an attractive alternative for the design of a new generation of stents.

Incorporation of fibronectin to enhance cytocompatibility in multilayer elastin‐like protein scaffolds for tissue engineering

Recombinant, elastin-like protein (ELP) polymers are of significant interest for the engineering of compliant, resilient soft tissues due to a wide range of tunable mechanical properties, biostability, and biocompatibility. Here, we enhance endothelial cell (EC) and mesenchymal stem cell compatibility with ELP constructs by addition of fibronectin (Fn) to the surface or bulk of ELP hydrogels. We find that cell adhesion, proliferation, and migration can be modulated by Fn addition. Adsorption of Fn to the hydrogel surface is more efficient than bulk blending. Surface immobilization of Fn by genipin crosslinking leads to stability without loss of bioactivity. Gels of varying mechanical modulus do not alter cell adhesion, proliferation, and migration in the range we investigate. However, more compliant gels promote an EC morphology suggesting tubulogenesis or network formation, whereas stiffer gels promote cobblestone morphology. Multilayer structures consisting of thin ELP sheets reinforced with collagen microfiber are fabricated and laminated through the culture of MSCs at layer interfaces. High cell viability in the resulting three-dimensional constructs suggests the applicability of Fn to the design of strong, resilient artificial blood vessels and other soft tissue replacements.

Immobilisation of linear and cyclic RGD-peptides on titanium surfaces and their impact on endothelial cell adhesion and proliferation

Functional coatings on titanium vascular stents and endosseous dental implants could probably enhance endothelial cell (EC) adhesion and activity with a shortening of the wound healing time and an increase of peri-implant angiogenesis during early bone formation. Therefore, the role of the structure of linear and cyclic cell adhesive peptides Arg-Gly-Asp (l-RGD and c-RGD) on differently pre-treated titanium (Ti) surfaces (untreated, silanised vs. functionalised with l- and c-RGD peptides) on EC cell coverage and proliferation was evaluated. After 24 h and after 3 d, surface coverage of adherent cells was quantified and an alamarBlue® proliferation assay was conducted. After 24 h, l-RGD modified surfaces showed a significantly better coverage of adhered cells than untreated titanium (p=0.01). Differences between l-RGD surfaces and silanised Ti (p=0.066) as well as between l-RGD and c-RGD surfaces (p=0.191) were not significant. After 3 d, c-RGD surfaces showed a significantly higher cell coverage than untreated Ti, silanised and l-RGD titanium surfaces (all p<0.0001). After 24 h, c-RGD modified surfaces showed significant higher cell proliferation compared to untreated Ti (p=0.003). However, there were no differences in proliferation between c-RGD and l-RGD (p=0.126) or c-RGD and silanised titanium (p=0.196). After 3 d, proliferation on c-RGD surfaces outranged significantly untreated titanium (p=0.004), silanised (p=0.001) and l-RGD surfaces (p=0.023), whereas no significant difference could be found between untreated Ti and l-RGD surfaces (p=0.54). According to these results, the biomimetic coating of c-RGD peptides on conventional titanium surfaces showed a positive effect on EC cell coverage and proliferation. We were able to show that modifications of titanium surfaces with c-RGD are a promising approach in promoting endothelial cell growth.

Concentration Polarization of Oxidative Modification of Low-Density Lipoproteins: Its Effect on Oxidative Modification of Low-Density Lipoprotein Uptake and Apoptosis of the Endothelial Cells

There is increasing evidence suggesting that oxidative modification of low-density lipoprotein (ox-LDL) in the vessel wall plays a crucial role in the initiation and progression of atherogenesis. The purpose of this study is to substantiate our hypothesis that concentration polarization of ox-LDL may also occur in the arterial system, which in turn can lead to enhanced endothelial cell (EC) apoptosis and induce atherogenesis. Using a parallel-plate flow chamber technique, ox-LDL uptake and apoptosis of the human ECs cultured on permeable or nonpermeable membranes were analyzed. The experimental results showed that 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine (DiI)-ox-LDL uptake by the ECs increased with increasing concentration of DiI-ox-LDL in the perfusion solution. The overall average value of DiI-ox-LDL uptake was approximately 20% higher for the permeable group than that for the nonpermeable group. The results also showed that ox-LDL induced ECs death and apoptosis in the permeable group were approximately 12% and 26% higher than those in the nonpermeable group, respectively. The results from the in vitro model study, therefore, support our hypothesis that concentration polarization of ox-LDLs may occur in the arterial system. In conclusion, the occurrence of ox-LDL concentration polarization could enhance ox-LDL infiltration into the arterial wall and accelerate EC apoptosis.

Abstract 3668: Endothelial Progenitor Cells Express Connexins which may be Important for the Functional Integration of Circulating Progenitor Cells into the Endothelium

Bone marrow (BM)-derived endothelial progenitor cells (EPC) enhance endothelial cell (EC) repair after focal EC damage, improve endothelial dysfunction, and are an important cellular risk predictor for cardiovascular mortality. For an effective EC repair, functional integration of circulating EPC into the damaged endothelium is a pre-requisite. Here, we determine the importance of connexins (Cx) for the interaction with mature ECs. Expression of Cx was evaluated on magnetic-bead selected CD133+ cells using immunocytochemistry and flow cytometry. CD133+ cells expressed Cx 40 and Cx 43 but not Cx 37. Mononuclear cells were cultivated to obtain early outgrowth EPC or colony forming units-endothelial cells (CFU-EC). Cx 37, 40, and 43 were expressed on cultured EPC. Co-culture experiments with mature human coronary artery endothelial cells (HCAEC) and EPC demonstrated the development of Cx 40, and 43 positive gap-junctions between both cells types. In order to investigate whether the interaction between HCAEC and EPC results in a functional communication between cells, dye transfer experiments were performed. Di-Ac-LDL labelled EPC transformed into Di-Ac-LDL and Calcein AM double positive cells when co-cultured with Calcein AM positive HCAEC and vice versa. In order to study the role of connexins in reendothelialization, C57bl6 (wild-type, WT) mice were subjected to a focal endothelial cell denudation and repetitively treated with WT and Cx37−/− deficient mononuclear cells. In contrast to the treatment with WT mononuclear cells, treatment with Cx37−/− cells was not associated with enhanced reendothelialization. Furthermore, endothelial function of isolated aortic rings derived from Cx37−/− mice showed an impaired endothelial-dependent vasorelaxation compared to WT mice. We provide evidence that connexins are differentially expressed on immature naíve EPC and cultured early-outgrowth EPC. The expression of connexin 37, 40, and 43 may be required for the functional integration of endothelial progenitor cells into the damaged endothelium.