Oncogene-induced matrix reorganization controls CD8+ T cell function in the soft-tissue sarcoma microenvironment.

CD8+ T cell dysfunction impedes anti-tumor immunity in solid cancers but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional co-activator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend upon oncogene-mediated ECM composition and remodeling.

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.

Cytoplasmic accumulation and plasma membrane association of anillin and Ect2 promote confined migration and invasion.

Cells migrating in confinement experience mechanical challenges whose consequences on cell migration machinery remain only partially understood. Here, we demonstrate that a pool of the cytokinesis regulatory protein anillin is retained during interphase in the cytoplasm of different cell types. Confinement induces recruitment of cytoplasmic anillin to plasma membrane at the poles of migrating cells, which is further enhanced upon nuclear envelope (NE) rupture(s). Rupture events also enable the cytoplasmic egress of predominantly nuclear RhoGEF Ect2. Anillin and Ect2 redistributions scale with microenvironmental stiffness and confinement, and are observed in confined cells in vitro and in invading tumor cells in vivo. Anillin, which binds actomyosin at the cell poles, and Ect2, which activates RhoA, cooperate additively to promote myosin II contractility, and promote efficient invasion and extravasation. Overall, our work provides a mechanistic understanding of how cytokinesis regulators mediate RhoA/ROCK/myosin II-dependent mechanoadaptation during confined migration and invasive cancer progression.

Hydrogels with tunable mechanical plasticity regulate endothelial cell outgrowth in vasculogenesis and angiogenesis.

The endothelial cell (EC) outgrowth in both vasculogenesis and angiogenesis starts with remodeling surrounding matrix and proceeds with the crosstalk between cells for the multicellular vasculature formation. The mechanical plasticity of matrix, defined as the ability to permanently deform by external traction, is pivotal in modulating cell behaviors. Nevertheless, the implications of matrix plasticity on cell-to-cell interactions during EC outgrowth, along with the molecular pathways involved, remain elusive. Here we develop a collagen-hyaluronic acid based hydrogel platform with tunable plasticity by using compositing strategy of dynamic and covalent networks. We show that although the increasing plasticity of the hydrogel facilitates the matrix remodeling by ECs, the largest tubular lumens and the longest invading distance unexpectedly appear in hydrogels with medium plasticity instead of the highest ones. We unravel that the high plasticity of the hydrogels promotes stable integrin cluster of ECs and recruitment of focal adhesion kinase with an overenhanced contractility which downregulates the vascular endothelial cadherin expression and destabilizes the adherens junctions between individual ECs. Our results, further validated with mathematical simulations and in vivo angiogenic tests, demonstrate that a balance of matrix plasticity facilitates both cell-matrix binding and cell-to-cell adherens, for promoting vascular assembly and invasion.

Downregulation of YAP Activity Restricts P53 Hyperactivation to Promote Cell Survival in Confinement.

Cell migration through confining three dimensional (3D) topographies can lead to loss of nuclear envelope integrity, DNA damage, and genomic instability. Despite these detrimental phenomena, cells transiently exposed to confinement do not usually die. Whether this is also true for cells subjected to long-term confinement remains unclear at present. To investigate this, photopatterning and microfluidics are employed to fabricate a high-throughput device that circumvents limitations of previous cell confinement models and enables prolonged culture of single cells in microchannels with physiologically relevant length scales. The results of this study show that continuous exposure to tight confinement can trigger frequent nuclear envelope rupture events, which in turn promote P53 activation and cell apoptosis. Migrating cells eventually adapt to confinement and evade cell death by downregulating YAP activity. Reduced YAP activity, which is the consequence of confinement-induced YAP1/2 translocation to the cytoplasm, suppresses the incidence of nuclear envelope rupture and abolishes P53-mediated cell death. Cumulatively, this work establishes advanced, high-throughput biomimetic models for better understanding cell behavior in health and disease, and underscores the critical role of topographical cues and mechanotransduction pathways in the regulation of cell life and death.

Collagen VI deposition mediates stromal T cell trapping through inhibition of T cell motility in the prostate tumor microenvironment.

The tumor extracellular matrix (ECM) is a barrier to anti-tumor immunity in solid tumors by disrupting T cell-tumor cell interaction underlying the need for elucidating mechanisms by which specific ECM proteins impact T cell motility and activity within the desmoplastic stroma of solid tumors. Here, we show that Collagen VI (Col VI) deposition correlates with stromal T cell density in human prostate cancer specimens. Furthermore, motility of CD4+ T cells is completely ablated on purified Col VI surfaces when compared with Fibronectin and Collagen I. Importantly, T cells adhered to Col VI surfaces displayed reduced cell spreading and fibrillar actin, indicating a reduction in traction force generation accompanied by a decrease in integrin ß1 clustering. We found that CD4+ T cells largely lack expression of integrin α1 in the prostate tumor microenvironment and that blockade of α1ß1 integrin heterodimers inhibited CD8+ T cell motility on prostate fibroblast-derived matrix, while re-expression of ITGA1 improved motility. Taken together, we show that the Col VI-rich microenvironment in prostate cancer reduces the motility of CD4+ T cells lacking integrin α1, leading to their accumulation in the stroma, thus putatively inhibiting anti-tumor T cell responses.

Hydrogels to Recapture Extracellular Matrix Cues That Regulate Vascularization.

The ECM (extracellular matrix) is a 3-dimensional network that supports cellular responses and maintains structural tissue integrity in healthy and pathological conditions. The interactions between ECM and cells trigger signaling cascades that lead to phenotypic changes and structural and compositional turnover of the ECM, which in turn regulates vascular cell behavior. Hydrogel biomaterials are a powerful platform for basic and translational studies and clinical applications due to their high swelling capacity and exceptional versatility in compositions and properties. This review highlights recent developments and uses of engineered natural hydrogel platforms that mimic the ECM and present defined biochemical and mechanical cues for vascularization. Specifically, we focus on modulating vascular cell stimulation and cell-ECM/cell-cell interactions in the microvasculature that are the established biomimetic microenvironment.

Vascular stiffening in aging females with a hypertension-induced HIF2A gain-of-function mutation.

Pulmonary arterial hypertension (PAH) is more prevalent in females than males; the causes of this sex difference have not been adequately explored. Gain-of-function (GOF) mutations in hypoxia-inducible factor 2α (HIF2A) lead to PAH and thrombotic consequences in patients and mice. Additionally, multiple emerging studies suggest that elevated systemic arterial stiffening (SAS) occurs in PAH; this could have critical prognostic value. Here, we utilized a HIF2A GOF mouse model to determine how SAS can be used as a prognosticator in sex-divergent PAH. We analyzed survival, vascular mechanics, and vascular phenotypes in young adult (8-16 weeks) and middle age (9-12 months) Hif2a GOF mice. We find that Hif2a heterozygous (HT) female mice, but not Hif2a HT male mice, exhibit poor survival, SAS upon aging, and decreased ability to withstand repeated physiological strain. Hif2a HT female mice also display thickening of the adventitial intima and increased collagen I and collagen III in all layers of the thoracic aorta. Our findings demonstrate differing PAH progression in female and male Hif2a GOF mice. Specifically, alterations in extracellular matrix (ECM) content led to vascular stiffening in aged females, resulting in poor survival. Moreover, we show that SAS emerges early in mice with PAH by coupling studies of vascular mechanics and analyzing vascular structure and composition. Importantly, we present a model for assessing sex differences in hereditary PAH progression and sex-specific prognosis, proposing that aortic stiffening can be used to prognosticate future poor outcomes in PAH.

BEYOND THE ENDOTHELIUM: THE ROLE OF MURAL CELLS IN VASCULAR BIOLOGY: In vitro systems to study endothelial/pericyte cell interactions.

The vasculature is crucial for tissue development and survival, and the stability of blood vessels to perform these functions relies on the interplay between endothelial cells (ECs) and mural cells. Pericytes are a subtype of mural cells found in the microvasculature that extend their processes to wrap around the endothelial monolayer. Pericytes are recruited during vessel growth through the excretion of soluble factors from ECs where they stabilize angiogenic sprouts and induce maturation of the resident cells. Alterations in these interactions between ECs and pericytes are associated with aberrant vessel growth and disrupted vasculature function characteristic of numerous diseases. Therefore, deeper understanding of the cross-talk between these cell types has numerous implications for understanding morphogenesis and elucidating disease mechanisms. In this review, we highlight recent advances and current trends studying the interactions between ECs and pericytes in vitro. We begin by analyzing three-dimensional hydrogel platforms that mimic the tissue extracellular matrix to investigate signaling pathways and altered vascular function in disease-specific cells. We next examine how microfluidic vasculature-on-a-chip platforms have elucidated the interplay of these vascular cells during angiogenesis and vascular network formation under controlled physiochemical cues and interstitial flow. Additionally, studies have utilized microvessels to measure the effect of shear stress on barrier function through the control of luminal flow and the impact of inflammation on these vascular cell interactions. Finally, we briefly highlight self-assembling human blood vessel organoids, an emerging high-throughput platform to study ECs and pericyte interactions.

Engineering of the microenvironment to accelerate vascular regeneration.

Blood vessels are crucial for tissue development, functionality, and homeostasis and are typically a determinant in the progression of healing and regeneration. The tissue microenvironment provides physicochemical cues that affect cellular function, and the study of the microenvironment can be accelerated by the engineering of approaches capable of mimicking various aspects of the microenvironment. In this review, we introduce the major components of the vascular niche and focus on the roles of oxygen and the extracellular matrix (ECM). We demonstrate how vascular engineering approaches enhance our understanding of the microenvironment's impact on the vasculature towards vascular regeneration and describe the current limitations and future directions towards clinical utilization.

Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

Nanoparticle-based modulation of CD4+ T cell effector and helper functions enhances adoptive immunotherapy.

Helper (CD4+) T cells perform direct therapeutic functions and augment responses of cells such as cytotoxic (CD8+) T cells against a wide variety of diseases and pathogens. Nevertheless, inefficient synthetic technologies for expansion of antigen-specific CD4+ T cells hinders consistency and scalability of CD4+ T cell-based therapies, and complicates mechanistic studies. Here we describe a nanoparticle platform for ex vivo CD4+ T cell culture that mimics antigen presenting cells (APC) through display of major histocompatibility class II (MHC II) molecules. When combined with soluble co-stimulation signals, MHC II artificial APCs (aAPCs) expand cognate murine CD4+ T cells, including rare endogenous subsets, to induce potent effector functions in vitro and in vivo. Moreover, MHC II aAPCs provide help signals that enhance antitumor function of aAPC-activated CD8+ T cells in a mouse tumor model. Lastly, human leukocyte antigen class II-based aAPCs expand rare subsets of functional, antigen-specific human CD4+ T cells. Overall, MHC II aAPCs provide a promising approach for harnessing targeted CD4+ T cell responses.

Engineering Smooth Muscle to Understand Extracellular Matrix Remodeling and Vascular Disease.

The vascular smooth muscle is vital for regulating blood pressure and maintaining cardiovascular health, and the resident smooth muscle cells (SMCs) in blood vessel walls rely on specific mechanical and biochemical signals to carry out these functions. Any slight change in their surrounding environment causes swift changes in their phenotype and secretory profile, leading to changes in the structure and functionality of vessel walls that cause pathological conditions. To adequately treat vascular diseases, it is essential to understand how SMCs crosstalk with their surrounding extracellular matrix (ECM). Here, we summarize in vivo and traditional in vitro studies of pathological vessel wall remodeling due to the SMC phenotype and, conversely, the SMC behavior in response to key ECM properties. We then analyze how three-dimensional tissue engineering approaches provide opportunities to model SMCs' response to specific stimuli in the human body. Additionally, we review how applying biomechanical forces and biochemical stimulation, such as pulsatile fluid flow and secreted factors from other cell types, allows us to study disease mechanisms. Overall, we propose that in vitro tissue engineering of human vascular smooth muscle can facilitate a better understanding of relevant cardiovascular diseases using high throughput experiments, thus potentially leading to therapeutics or treatments to be tested in the future.

Off-the-shelf, heparinized small diameter vascular graft limits acute thrombogenicity in a porcine model.

Thrombogenicity poses a challenge to the clinical translation of engineered grafts. Previously, small-diameter vascular grafts (sdVG) composed of fibrin hydrogel microfiber tubes (FMT) with an external poly(ε-caprolactone) (PCL) sheath supported long-term patency in mice. Towards the development of an sdVG with off-the-shelf availability, the FMT's shelf stability, scale-up, and successful conjugation of an antithrombotic drug to the fibrin scaffold are reported here. FMTs maintain mechanical stability and high-water retention after storage for one year in a freezer, in a refrigerator, or at room temperature. Low molecular weight heparin-conjugated fibrin scaffolds enabled local and sustained delivery during two weeks of enzymatic degradation. Upscaled fabrication of sdVGs provides natural biodegradable grafts with size and mechanics suitable for human application. Implantation in a carotid artery interposition porcine model exhibited no rupture with thrombi prevented in all heparinized sdVGs (n = 4) over 4-5 weeks. Remodeling of the sdVGs is demonstrated with endothelial cells on the luminal surface and initial formation of the medial layer by 4-5 weeks. However, neointimal hyperplasia at 4-5 weeks led to the stenosis and occlusion of most of the sdVGs, which must be resolved for future long-term in vivo assessments. The off-the-shelf, biodegradable heparinized fibrin sdVG layer limits acute thrombogenicity while mediating extensive neotissue formation as the PCL sheath maintains structural integrity. STATEMENT OF SIGNIFICANCE: To achieve clinical and commercial utility of small-diameter vascular grafts as arterial conduits, these devices must have off-the-shelf availability for emergency arterial bypass applications and be scaled to a size suitable for human applications. A serious impediment to clinical translation is thrombogenicity. Treatments have focused on long-term systemic drug therapy, which increases the patient's risk of bleeding complications, or coating grafts and stents with anti-coagulants, which minimally improves patient outcomes even when combined with dual anti-platelet therapy. We systematically modified the biomaterial properties to develop anticoagulant embedded, biodegradable grafts that maintain off-the-shelf availability, provide mechanical stability, and prevent clot formation through local drug delivery.

Tissue Engineering Approaches to Uncover Therapeutic Targets for Endothelial Dysfunction in Pathological Microenvironments.

Endothelial cell dysfunction plays a central role in many pathologies, rendering it crucial to understand the underlying mechanism for potential therapeutics. Tissue engineering offers opportunities for in vitro studies of endothelial dysfunction in pathological mimicry environments. Here, we begin by analyzing hydrogel biomaterials as a platform for understanding the roles of the extracellular matrix and hypoxia in vascular formation. We next examine how three-dimensional bioprinting has been applied to recapitulate healthy and diseased tissue constructs in a highly controllable and patient-specific manner. Similarly, studies have utilized organs-on-a-chip technology to understand endothelial dysfunction's contribution to pathologies in tissue-specific cellular components under well-controlled physicochemical cues. Finally, we consider studies using the in vitro construction of multicellular blood vessels, termed tissue-engineered blood vessels, and the spontaneous assembly of microvascular networks in organoids to delineate pathological endothelial dysfunction.

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.

Intrinsic epigenetic control of angiogenesis in induced pluripotent stem cell-derived endothelium regulates vascular regeneration.

Human-induced pluripotent stem cell-derived endothelial cells (iECs) provide opportunities to study vascular development and regeneration, develop cardiovascular therapeutics, and engineer model systems for drug screening. The differentiation and characterization of iECs are well established; however, the mechanisms governing their angiogenic phenotype remain unknown. Here, we aimed to determine the angiogenic phenotype of iECs and the regulatory mechanism controlling their regenerative capacity. In a comparative study with HUVECs, we show that iECs increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) mediates their highly angiogenic phenotype via regulation of glycolysis enzymes, filopodia formation, VEGF mediated migration, and robust sprouting. We find that the elevated expression of VEGFR2 is epigenetically regulated via intrinsic acetylation of histone 3 at lysine 27 by histone acetyltransferase P300. Utilizing a zebrafish xenograft model, we demonstrate that the ability of iECs to promote the regeneration of the amputated fin can be modulated by P300 activity. These findings demonstrate how the innate epigenetic status of iECs regulates their phenotype with implications for their therapeutic potential.

Hypoxia-induced blood-brain barrier dysfunction is prevented by pericyte-conditioned media via attenuated actomyosin contractility and claudin-5 stabilization.

The blood-brain barrier (BBB) regulates molecular and cellular entry from the cerebrovasculature into the surrounding brain parenchyma. Many diseases of the brain are associated with dysfunction of the BBB, where hypoxia is a common stressor. However, the contribution of hypoxia to BBB dysfunction is challenging to study due to the complexity of the brain microenvironment. In this study, we used a BBB model with brain microvascular endothelial cells and pericytes differentiated from iPSCs to investigate the effect of hypoxia on barrier function. We found that hypoxia-induced barrier dysfunction is dependent upon increased actomyosin contractility and is associated with increased fibronectin fibrillogenesis. We propose a role for actomyosin contractility in mediating hypoxia-induced barrier dysfunction through modulation of junctional claudin-5. Our findings suggest pericytes may protect brain microvascular endothelial cells from hypoxic stresses and that pericyte-derived factors could be candidates for treatment of pathological barrier-forming tissues.

Discretizing Three-Dimensional Oxygen Gradients to Modulate and Investigate Cellular Processes.

With the increased realization of the effect of oxygen (O2 ) deprivation (hypoxia) on cellular processes, recent efforts have focused on the development of engineered systems to control O2 concentrations and establish biomimetic O2 gradients to study and manipulate cellular behavior. Nonetheless, O2 gradients present in 3D engineered platforms result in diverse cell behavior across the O2 gradient, making it difficult to identify and study O2 sensitive signaling pathways. Using a layer-by-layer assembled O2 -controllable hydrogel, the authors precisely control O2 concentrations and study uniform cell behavior in discretized O2 gradients, then recapitulate the dynamics of cluster-based vasculogenesis, one mechanism for neovessel formation, and show distinctive gene expression patterns remarkably correlate to O2 concentrations. Using RNA sequencing, it is found that time-dependent regulation of cyclic adenosine monophosphate signaling enables cell survival and clustering in the high stress microenvironments. Various extracellular matrix modulators orchestrate hypoxia-driven endothelial cell clustering. Finally, clustering is facilitated by regulators of cell-cell interactions, mainly vascular cell adhesion molecule 1. Taken together, novel regulators of hypoxic cluster-based vasculogenesis are identified, and evidence for the utility of a unique platform is provided to study dynamic cellular responses to 3D hypoxic environments, with broad applicability in development, regeneration, and disease.