Age-dependent loss of HAPLN1 erodes vascular integrity via indirect upregulation of endothelial ICAM1 in melanoma.

Melanoma, the most lethal form of skin cancer, often has worse outcomes in older patients. We previously demonstrated that an age-related decrease in the secreted extracellular matrix (ECM) protein HAPLN1 has a role in slowing melanoma progression. Here we show that HAPLN1 in the dermal ECM is sufficient to maintain the integrity of melanoma-associated blood vessels, as indicated by increased collagen and VE-cadherin expression. Specifically, we show that HAPLN1 in the ECM increases hyaluronic acid and decreases endothelial cell expression of ICAM1. ICAM1 phosphorylates and internalizes VE-cadherin, a critical determinant of vascular integrity, resulting in permeable blood vessels. We found that blocking ICAM1 reduces tumor size and metastasis in older mice. These results suggest that HAPLN1 alters endothelial ICAM1expression in an indirect, matrix-dependent manner. Targeting ICAM1 could be a potential treatment strategy for older patients with melanoma, emphasizing the role of aging in tumorigenesis.

Stiffening Matrix Induces Age-Mediated Microvascular Phenotype Through Increased Cell Contractility and Destabilization of Adherens Junctions.

Aging is a major risk factor in microvascular dysfunction and disease development, but the underlying mechanism remains largely unknown. As a result, age-mediated changes in the mechanical properties of tissue collagen have gained interest as drivers of endothelial cell (EC) dysfunction. 3D culture models that mimic age-mediated changes in the microvasculature can facilitate mechanistic understanding. A fibrillar hydrogel capable of changing its stiffness after forming microvascular networks is established. This hydrogel model is used to form vascular networks from induced pluripotent stem cells under soft conditions that mimic young tissue mechanics. Then matrix stiffness is gradually increased, thus exposing the vascular networks to the aging-mimicry process in vitro. It is found that upon dynamic matrix stiffening, EC contractility is increased, resulting in the activation of focal adhesion kinase and subsequent dissociation of ß-catenin from VE-Cadherin mediated adherens junctions, leading to the abruption of the vascular networks. Inhibiting cell contractility impedes the dissociation of ß-catenin, thereby preventing the deconstruction of adherens junctions, thus partially rescuing the age-mediated vascular phenotype. The findings provide the first direct evidence of matrix's dynamic mechano-changes in compromising microvasculature with aging and highlight the importance of hydrogel systems to study tissue-level changes with aging in basic and translational studies.

Advances in ADAMTS biomarkers.

A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) are major mediators in extracellular matrix (ECM) turnover and have gained increasing interest over the last years as major players in ECM remodeling during tissue homeostasis and the development of diseases. Although, ADAMTSs are recognized in playing important roles during tissue remodeling, and loss of function in various member of the ADAMTS family could be associated with the development of numerous diseases, limited knowledge is available about their specific substrates and mechanism of action. In this chapter, we will review current knowledge about ADAMTSs and their use as disease biomarkers.

Hydrogel Network Dynamics Regulate Vascular Morphogenesis.

Matrix dynamics influence how individual cells develop into complex multicellular tissues. Here, we develop hydrogels with identical polymer components but different crosslinking capacities to enable the investigation of mechanisms underlying vascular morphogenesis. We show that dynamic (D) hydrogels increase the contractility of human endothelial colony-forming cells (hECFCs), promote the clustering of integrin ß1, and promote the recruitment of vinculin, leading to the activation of focal adhesion kinase (FAK) and metalloproteinase expression. This leads to the robust assembly of vasculature and the deposition of new basement membrane. We also show that non-dynamic (N) hydrogels do not promote FAK signaling and that stiff D- and N-hydrogels are constrained for vascular morphogenesis. Furthermore, D-hydrogels promote hECFC microvessel formation and angiogenesis in vivo. Our results indicate that cell contractility mediates integrin signaling via inside-out signaling and emphasizes the importance of matrix dynamics in vascular tissue formation, thus informing future studies of vascularization and tissue engineering applications.

Preparation and Application of a Decellularized Extracellular Matrix for Identification of ADAMTS Substrates.

Here we describe the use of a decellularized ECM produced in vitro by BALB/c 3T3 fibroblasts for the identification of ADAMTS substrates. Seeding of ADAMTS protease-producing HEK cells on top of the cell-free ECM followed by analysis of the conditioned medium by liquid chromatography tandem mass spectrometry (LC-MS/MS), allows for screening of ADAMTS substrates without prior purification of full-length protease.

A Selective Extracellular Matrix Proteomics Approach Identifies Fibronectin Proteolysis by A Disintegrin-like and Metalloprotease Domain with Thrombospondin Type 1 Motifs (ADAMTS16) and Its Impact on Spheroid Morphogenesis.

Secreted and cell-surface proteases are major mediators of extracellular matrix (ECM) turnover, but their mechanisms and regulatory impact are poorly understood. We developed a mass spectrometry approach using a cell-free ECM produced in vitro to identify fibronectin (FN) as a novel substrate of the secreted metalloprotease ADAMTS16. ADAMTS16 cleaves FN between its (I)5 and (I)6 modules, releasing the N-terminal 30 kDa heparin-binding domain essential for FN self-assembly. ADAMTS16 impairs FN fibrillogenesis as well as fibrillin-1 and tenascin-C assembly, thus inhibiting formation of a mature ECM by cultured fibroblasts. Furthermore ADAMTS16 has a marked morphogenetic impact on spheroid formation by renal tubule-derived MDCKI cells. The N-terminal FN domain released by ADAMTS16 up-regulates MMP3, which cleaves the (I)5-(I)6 linker of FN similar to ADAMTS16, therefore creating a proteolytic feed-forward mechanism. Thus, FN proteolysis not only regulates FN turnover, but also FN assembly, with potential long-term consequences for ECM assembly and morphogenesis.