Loss of endothelial glucocorticoid receptor promotes angiogenesis via upregulation of Wnt/ß-catenin pathway.

OBJECTIVE: The glucocorticoid receptor (GR) is a member of the nuclear receptor family that controls key biological processes in the cardiovascular system and has recently been shown to modulate Wnt signaling in endothelial cells. Wnt/ß-catenin signaling has been demonstrated to be crucial in the process of angiogenesis. In the current study, we studied whether GR could regulate angiogenesis via the Wnt/ß-catenin pathway. APPROACH AND RESULTSA: Key components of the Wnt/ß-catenin pathway were evaluated using quantitative PCR and Western blot in the presence or absence of GR. Enhanced angiogenesis was found in GR deficiency in vitro and confirmed with cell viability assays, proliferation assays and tube formation assays. Consistent with these in vitro findings, endothelial cell-specific GR loss GR in vivo promoted angiogenesis in both a hind limb ischemia model and sponge implantation assay. Results were further verified in a novel mouse model lacking endothelial LRP5/6, a key receptor in canonical Wnt signaling, and showed substantially suppressed angiogenesis using these same in vitro and in vivo assays. To further investigate the mechanism of GR regulation of Wnt signaling, autophagy flux was investigated in endothelial cells by visualizing auto phagolysosomes as well as by assessing P62 degradation and LC3B conversion. Results indicated that potentiated autophagy flux participated in GR-Wnt regulation. CONCLUSIONS: Lack of endothelial GR triggers autophagy flux, leads to activation of Wnt/ß-catenin signaling and promotes angiogenesis. There may also be a synergistic interaction between autophagy and Wnt/ß-catenin signaling.

An in situ collagen-HA hydrogel system promotes survival and preserves the proangiogenic secretion of hiPSC-derived vascular smooth muscle cells.

Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration.

Endothelial Cell Autonomous Role of Akt1: Regulation of Vascular Tone and Ischemia-Induced Arteriogenesis.

OBJECTIVE: The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. APPROACH AND RESULTS: Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. CONCLUSIONS: Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.

Impaired von Willebrand factor adhesion and platelet response in thrombospondin-2 knockout mice.

Interactions between collagenous extracellular matrices and von Willebrand factor (VWF) are critical for hemostasis and thrombosis. In the present study, we investigated the contribution of an extracellular matrix (ECM) abnormality to the bleeding diathesis in thrombospondin-2 (TSP2) knockout (KO) mice. First, we performed adoptive bone marrow transplantation and observed that introduction of wild-type (WT) marrow into lethally irradiated TSP2 KO mice did not rescue the bleeding diathesis. However, platelets in transplanted mice displayed an inherent aggregation defect, which complicated interpretation. Second, we performed interposition of arterial segments denuded of endothelium. Denuded TSP2 KO arteries grafted into WT mice remained patent in vivo. In contrast, WT grafts underwent thrombosis and were completely occluded within 24 to 48 hours. The nonthrombogenic property of the TSP2 KO ECM was confirmed in vitro by exposing platelets to TSP2 KO dermal fibroblast (DF)-derived ECM. To further probe the effect of TSP2 deficiency, ECM production and deposition by WT and TSP2 KO DFs was analyzed via polymerase chain reaction, immunofluorescence, and scanning electron microscopy and showed similar patterns. In addition, atomic force microscopy (AFM) analysis of WT and TSP2 KO ECM did not reveal differences in stiffness. In contrast, reduced VWF accumulation on TSP2 KO ECM was observed when matrices were subjected to plasma under physiological flow. AFM utilizing VWF-coated 2-µm beads confirmed the weak binding to TSP2 KO ECM, providing a mechanistic explanation for the lack of thrombus formation. Therefore, our studies show that ECM assembly is critical for interaction of collagen with VWF and subsequent thrombogenic responses.

New Functional Tools for Antithrombogenic Activity Assessment of Live Surface Glycocalyx.

OBJECTIVE: It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in many vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators' ability to assess the role of the glycocalyx in vascular health. APPROACH AND RESULTS: We have developed novel, easy-to-use, in vitro assays that directly quantify live endothelialized surface's functional heparin weights and their anticoagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized 2 commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell monolayer. We determined heparin contents to be ≈10 000 ng/cm(2) on the native aorta and ≈10-fold lower on cultured human umbilical vein endothelial cells. Interestingly, human umbilical vein endothelial cells demonstrated a 5-fold lower anticoagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using liquid chromatography-mass spectrometry analysis. CONCLUSIONS: Our assays are of high relevance in the vascular community because they can be used to establish the antithrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.

Endothelial Akt1 mediates angiogenesis by phosphorylating multiple angiogenic substrates.

The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas nonphysiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akt1-deficient, but not Akt2-deficient, ECs, including eNOS and Forkhead box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling.

Up-regulation of thrombospondin-2 in Akt1-null mice contributes to compromised tissue repair due to abnormalities in fibroblast function.

Vascular remodeling is essential for tissue repair and is regulated by multiple factors, including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knock-out (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full-thickness excisional wounds in DKO mice healed at an accelerated rate when compared with Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, the addition of exogenous TSP2 to WT cells induced cell morphology and migration rates that were similar to those of Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared with WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions; however, this effect was not due to changes in ß1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair.

Thrombospondin-2 and extracellular matrix assembly.

BACKGROUND: Numerous proteins and small leucine-rich proteoglycans (SLRPs) make up the composition of the extracellular matrix (ECM). Assembly of individual fibrillar components in the ECM, such as collagen, elastin, and fibronectin, is understood at the molecular level. In contrast, the incorporation of non-fibrillar components and their functions in the ECM are not fully understood. SCOPE OF REVIEW: This review will focus on the role of the matricellular protein thrombospondin (TSP) 2 in ECM assembly. Based on findings in TSP2-null mice and in vitro studies, we describe the participation of TSP2 in ECM assembly, cell-ECM interactions, and modulation of the levels of matrix metalloproteinases (MMPs). MAJOR CONCLUSIONS: Evidence summarized in this review suggests that TSP2 can influence collagen fibrillogenesis without being an integral component of fibrils. Altered ECM assembly and excessive breakdown of ECM can have both positive and negative consequences including increased angiogenesis during tissue repair and compromised cardiac tissue integrity, respectively. GENERAL SIGNIFICANCE: Proper ECM assembly is critical for maintaining cell functions and providing structural support. Lack of TSP2 is associated with increased angiogenesis, in part, due to altered endothelial cell-ECM interactions. Therefore, minor changes in ECM composition can have profound effects on cell and tissue function. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Molecular Characterization of Macrophage-Biomaterial Interactions.

Implantation of biomaterials in vascularized tissues elicits the sequential engagement of molecular and cellular elements that constitute the foreign body response. Initial events include the non-specific adsorption of proteins to the biomaterial surface that render it adhesive for cells such as neutrophils and macrophages. The latter undergo unique activation and in some cases undergo cell-cell fusion to form foreign body giant cells that contribute to implant damage and fibrotic encapsulation. In this review, we discuss the molecular events that contribute to macrophage activation and fusion with a focus on the role of the inflammasome, signaling pathways such as JAK/STAT and NF-κB, and the putative involvement of micro RNAs in the regulation of these processes.

Endothelial nitric oxide synthase controls the expression of the angiogenesis inhibitor thrombospondin 2.

Injury- and ischemia-induced angiogenesis is critical for tissue repair and requires nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS). We present evidence that NO induces angiogenesis by modulating the level of the angiogenesis inhibitor thrombospondin 2 (TSP2). TSP2 levels were higher than WT in eNOS KO tissues in hind-limb ischemia and cutaneous wounds. In vitro studies confirmed that NO represses TSP2 promoter activity. Moreover, double-eNOS/TSP2 KO mice were generated and found to rescue the phenotype of eNOS KO mice. Studies in mice with knock-in constitutively active or inactive eNOS on the Akt-1 KO background showed that eNOS activity correlates with TSP2 levels. Our observations of NO-mediated regulation of angiogenesis via the suppression of TSP2 expression provide a description of improved eNOS KO phenotype by means other than restoring NO signaling.

Inflammasome components Asc and caspase-1 mediate biomaterial-induced inflammation and foreign body response.

Implantation of biomaterials and devices into soft tissues leads to the development of the foreign body response (FBR), which can interfere with implant function and eventually lead to failure. The FBR consists of overlapping acute and persistent inflammatory phases coupled with collagenous encapsulation and currently there are no therapeutic options. Initiation of the FBR involves macrophage activation, proceeding to giant cell formation, fibroblast activation, and collagen matrix deposition. Despite the recognition of this sequence of events, the molecular pathways required for the FBR have not been elucidated. We have identified that the acute inflammatory response to biomaterials requires nucleotide-binding domain and leucine-rich repeat-containing 3 (Nlrp3), apoptosis-associated speck-like protein containing CARD (Asc), and caspase-1, as well as plasma membrane cholesterol, and Syk signaling. Full development of the FBR is dependent on Asc and caspase-1, but not Nlrp3. The common antiinflammatory drug aspirin can reduce inflammasome activation and significantly reduce the FBR. Taken together, these findings expand the role of the inflammasome from one of sensing damage associated molecular patterns (DAMPs) to sensing all particulate matter irrespective of size. In addition, implication of the inflammasome in biomaterial recognition identifies key pathways, which can be targeted to limit the FBR.

Novel muscle-derived extracellular matrix hydrogel promotes angiogenesis and neurogenesis in volumetric muscle loss.

Volumetric muscle loss (VML) represents a clinical challenge due to the limited regenerative capacity of skeletal muscle. Most often, it results in scar tissue formation and loss of function, which cannot be prevented by current therapies. Decellularized extracellular matrix (DEM) has emerged as a native biomaterial for the enhancement of tissue repair. Here, we report the generation and characterization of hydrogels derived from DEM prepared from WT or thrombospondin (TSP)-2 null muscle tissue. TSP2-null hydrogels, when compared to WT, displayed altered architecture, protein composition, and biomechanical properties and allowed enhanced invasion of C2C12 myocytes and chord formation by endothelial cells. They also displayed enhanced cell invasion, innervation, and angiogenesis following subcutaneous implantation. To evaluate their regenerative capacity, WT or TSP2 null hydrogels were used to treat VML injury to tibialis anterior muscles and the latter induced greater recruitment of repair cells, innervation, and blood vessel formation and reduced inflammation. Taken together, these observations indicate that TSP2-null hydrogels enhance angiogenesis and promote muscle repair in a VML model.

Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.

Astrocyte-derived thrombospondin-2 is critical for the repair of the blood-brain barrier.

Thrombospondin (TSP)-2-null mice have an altered brain foreign body response (FBR) characterized by increases in inflammation, extracellular matrix deposition, and leakage of the blood-brain barrier (BBB). In the present study, we investigated the role of TSP-2 in BBB repair during the brain FBR to mixed cellulose ester filters implanted in the cortex of wild-type (WT) and TSP-2-null mice for 2 days to 8 weeks. Histological and immunohistochemical analysis revealed enhanced and prolonged neuroinflammation in TSP-2-null mice up to 8 weeks after implantation. In addition, recovery of the BBB was compromised and was associated with increased gelatinolytic activity and low levels of collagen type IV in the basement membranes of TSP-2-null blood vessels. An analysis of protein extracts from implantation sites revealed elevated levels of matrix metalloproteinase (MMP)-2 and MMP-9 in TSP-2-null brains. TSP-2-null astrocytes secreted higher levels of both MMPs in vitro compared with their WT counterparts. Furthermore, TSP-2-null astrocytes were deficient in supporting the recovery of barrier function in WT endothelial cells. Finally, Western blot analysis of astrocytes and brain endothelial cells revealed TSP-2 expression only in the former. Taken together, our observations suggest that astrocyte-derived TSP-2 is critical for the maintenance of physiological MMP-2 and MMP-9 levels during the FBR and contributes to the repair of the BBB.

Lack of TNF-α-induced MMP-9 production and abnormal E-cadherin redistribution associated with compromised fusion in MCP-1-null macrophages.

Homotypic cell fusion occurs in several cell types including macrophages in the formation of foreign body giant cells. Previously, monocyte chemoattractant protein-1 (MCP-1) was demonstrated to be required for foreign body giant cell formation in the foreign body response. The present study investigated the fusion defect in MCP-1-null macrophages by implanting biomaterials intraperitoneally in wild-type and MCP-1-null mice and monitoring the macrophage response at 12 hours to 4 weeks. MCP-1-null mice exhibited reduced accumulation and fusion of macrophages on implants, which was associated with attenuation of the foreign body response. Consistent with previous in vitro findings, the level of matrix metalloproteinase-9 (MMP-9) was reduced in MCP-1-null macrophages adherent to implants. In contrast, CCR2 expression was unaffected. In vitro studies revealed reduced tumor necrosis factor-α (TNF-α) production and abnormal subcellular redistribution of E-cadherin and ß-catenin during fusion in MCP-1-null macrophages. Exogenous TNF-α caused an increase in the production of MMP-9 and rescued the fusion defect. Addition of GM6001 (MMP inhibitor) or NSC23766 (Rac1 inhibitor) indicated two distinct induction pathways, one for E-cadherin/ß-catenin and one for MCP-1, TNF-α, and MMP-9. Considered together, these observations demonstrate that induction of E-cadherin/ß-catenin is not sufficient for fusion in the absence of MCP-1 or the downstream mediators TNF-α and MMP-9. Moreover, attenuation of the foreign body response in intraperitoneal implants in MCP-1-null mice demonstrates that the process depends on tissue-specific factors.

Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair.

Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control.

Foreign body response to synthetic polymer biomaterials and the role of adaptive immunity.

Implanted biomaterials elicit a series of distinct immune and repair-like responses that are collectively known as the foreign body reaction (FBR). These include processes involving innate immune inflammatory cells and wound repair cells that contribute to the encapsulation of biomaterials with a dense collagenous and largely avascular capsule. Numerous studies have shown that the early phase is dominated by macrophages that fuse to form foreign body giant cells that are considered a hallmark of the FBR. With the advent of more precise cell characterization techniques, specific macrophage subsets have been identified and linked to more or less favorable outcomes. Moreover, studies comparing synthetic- and natural-based polymer biomaterials have allowed the identification of macrophage subtypes that distinguish between fibrotic and regenerative responses. More recently, cells associated with adaptive immunity have been shown to participate in the FBR to synthetic polymers. This suggests the existence of cross-talk between innate and adaptive immune cells that depends on the nature of the implants. However, the exact participation of adaptive immune cells, such as T and B cells, remains unclear. In fact, contradictory studies suggest either the independence or dependence of the FBR on these cells. Here, we review the evidence for the involvement of adaptive immunity in the FBR to synthetic polymers with a focus on cellular and molecular components. In addition, we examine the possibility that such biomaterials induce specific antibody responses resulting in the engagement of adaptive immune cells.

Treating 'Septic' With Enhanced Antibiotics and 'Arthritis' by Mitigation of Excessive Inflammation.

Bacterial infection within the synovial joint, commonly known as septic arthritis, remains a clinical challenge as it presents two concurrent therapeutic goals of reducing bacterial burden and preservation of articular cartilage from destructive host inflammation. We hypothesized that mitigation of MRSA-induced inflammatory signaling could diminish destruction of articular cartilage in the setting of septic arthritis when used in conjunction with antibiotics. Herein, we provide evidence which supports a new therapeutic notion that concurrent antimicrobial therapy to address the 'septic' component of the disease with inflammation mitigation to manage the destructive 'arthritis' component. We established a murine model to mimic septic knee arthritis, as well as a variety of other inflammatory joint conditions. This murine septic arthritis model, in conjunction with in vitro and ex-vivo models, was utilized to characterize the inflammatory profile seen in active septic arthritis, as well as post-antibiotic treatment, via transcriptomic and histologic studies. Finally, we provided the clinical rationale for a novel therapeutic strategy combining enhanced antibiotic treatment with rifampin and adjuvant immunomodulation to inhibit post-infectious, excess chondrolysis and osteolysis. We identified that septic arthritis secondary to MRSA infection in our murine model led to increased articular cartilage damage compared to various types of inflammatory arthritis. The activation of the pERK1/2 signaling pathway, which is implicated with the mounting of an immune response and generation of inflammation, was increased in intracellular MRSA-infected synovial tissue and persisted despite antibiotic treatment. Trametinib, an inhibitor of ERK signaling through suppression of MEK1/2, alleviated the inflammation produced by the addition of intra-articular, heat-killed MRSA. Further, when combined with vancomycin and rifampin, mitigation of inflammation by pERK1/2 targeting improved outcomes for MRSA septic arthritis by conferring chondroprotection to articular cartilage and diminishing inflammatory osteolysis within bone. Our results support a new therapeutic notion that cell/biofilm-penetrating antibiotics alongside adjuvant mitigation of excessive intra-articular inflammation accomplish distinct therapeutic goals: reduction of bacterial burden and preservation of articular cartilage integrity.

Dysregulation of TSP2-Rac1-WAVE2 axis in diabetic cells leads to cytoskeletal disorganization, increased cell stiffness, and dysfunction.

Fibroblasts are a major cell population that perform critical functions in the wound healing process. In response to injury, they proliferate and migrate into the wound space, engaging in extracellular matrix (ECM) production, remodeling, and contraction. However, there is limited knowledge of how fibroblast functions are altered in diabetes. To address this gap, several state-of-the-art microscopy techniques were employed to investigate morphology, migration, ECM production, 2D traction, 3D contraction, and cell stiffness. Analysis of cell-derived matrix (CDM) revealed that diabetic fibroblasts produce thickened and less porous ECM that hindered migration of normal fibroblasts. In addition, diabetic fibroblasts were found to lose spindle-like shape, migrate slower, generate less traction force, exert limited 3D contractility, and have increased cell stiffness. These changes were due, in part, to a decreased level of active Rac1 and a lack of co-localization between F-actin and Waskott-Aldrich syndrome protein family verprolin homologous protein 2 (WAVE2). Interestingly, deletion of thrombospondin-2 (TSP2) in diabetic fibroblasts rescued these phenotypes and restored normal levels of active Rac1 and WAVE2-F-actin co-localization. These results provide a comprehensive view of the extent of diabetic fibroblast dysfunction, highlighting the regulatory role of the TSP2-Rac1-WAVE2-actin axis, and describing a new function of TSP2 in regulating cytoskeleton organization.

Integrin ß3 targeting biomaterial preferentially promotes secretion of bFGF and viability of iPSC-derived vascular smooth muscle cells.

Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) and their secretome have been shown to promote angiogenesis and wound healing. However, there is a paucity of research on how the extracellular matrix (ECM) microenvironment may impact the hiPSC-VSMC's functions. In this study, we investigated the effect of specific ECM ligand-integrin interaction on hiPSC-VSMC's paracrine secretion, cell viability, and morphology. Here, we show precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin ß3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin-dependent via αvß3 integrin interactions. In addition, our data show the possible role of a positive feedback loop between integrin ß3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC-VSMC's morphology and cell viability. Finally, the secretome with enhanced bFGF shows potential for future wound healing applications.