TGF-ß1 Drives Integrin-Dependent Pericyte Migration and Microvascular Destabilization in Fibrotic Disease.

Interactions between endothelial cells (ECs) and mural pericytes (PCs) are critical in maintaining the stability and function of the microvascular wall. Abnormal interactions between these two cell types are a hallmark of progressive fibrotic diseases such as systemic sclerosis (also known as scleroderma). However, the role of PCs in signaling microvascular dysfunction remains underexplored. We hypothesized that integrin-matrix interactions contribute to PC migration from the vascular wall and conversion into interstitial myofibroblasts. Herein, pro-inflammatory tumor necrosis factor α (TNFα) or a fibrotic growth factor [transforming growth factor ß1 (TGF-ß1)] were used to evaluate human PC inflammatory and fibrotic phenotypes by assessing their migration, matrix deposition, integrin expression, and subsequent effects on endothelial dysfunction. Both TNFα and TGF-ß1 treatment altered integrin expression and matrix protein deposition, but only fibrotic TGF-ß1 drove PC migration in an integrin-dependent manner. In addition, integrin-dependent PC migration was correlated to changes in EC angiopoietin-2 levels, a marker of vascular instability. Finally, there was evidence of changes in vascular stability corresponding to disease state in human systemic sclerosis skin. This work shows that TNFα and TGF-ß1 induce changes in PC integrin expression and matrix deposition that facilitate migration and reduce vascular stability, providing evidence that microvascular destabilization can be an early indicator of tissue fibrosis.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

Endothelial cell secreted VEGF-C enhances NSC VEGFR3 expression and promotes NSC survival.

Although delivery of neural stem cell (NSC) as a therapeutic treatment for intracerebral hemorrhage (ICH) provides promise, NSC delivery typically has extremely low survival rates. Here, we investigate endothelial cell (EC) and pericyte (PC) interactions with NSC, where our results demonstrate that EC, and not PC, promote NSC cell proliferation and reduce cytotoxicity under glucose deprivation (GD). Additionally, NSC proliferation was increased upon treatment with EC conditioned media, inhibited with antagonism of VEGFR3. In an NSC + EC co-culture we detected elevated levels of VEGF-C, not seen for NSC cultured alone. Exogenous VEGF-C induced NSC upregulation of VEGFR3, promoted proliferation, and reduced cytotoxicity. Finally, we delivered microbeads containing NSC + EC into a murine ICH cavity, where VEGF-C was increasingly present in the injury site, not seen upon delivery NSC encapsulated alone. These studies demonstrate that EC-secreted VEGF-C may promote NSC survival during injury, enhancing the potential for cell delivery therapies for stroke.

Endothelial cell secreted metalloproteinase-2 enhances neural stem cell N-cadherin expression, clustering, and migration.

Neuroblasts have a clustered phenotype critical for their unidirectional migration, which in part is dependent on signaling from microvascular endothelial cells (EC) and pericytes (PC). Diffusible signals secreted by vascular cells have been demonstrated to increase survival, proliferation, and differentiation of subventricular zone resident neural stem cells (NSC); however, the signals that promote the necessary initiating step of NSC clustering are undefined. To investigate the role of vascular cells in promoting NSC clustering and directing migration, we created a 3-D hydrogel that mimics the biomechanics, biochemistry, and architectural complexity of brain tissue. We demonstrate that EC, and not PC, have a crucial role in NSC clustering and migration, further verified through microfluidic chamber systems and traction force microscopy. Ablation of the extended NSC aggregate arm halts aggregate movement, suggesting that clustering is a prerequisite for migration. When cultured with EC, NSC clustering occurs and NSC coincidentally increase their expression of N-cadherin, as compared to NSC cultured alone. NSC-presented N-cadherin expression was increased following exposure to EC secreted metalloproteinase-2 (MMP2). We demonstrate that inhibition of MMP2 prevented NSC N-cadherin surface expression and subsequent NSC clustering, even when NSC were in direct contact with EC. Furthermore, with exogenous activation of EGFR, which serves as a downstream activator of N-cadherin cleavage, NSC form clusters. Our results suggest that EC secretion of MMP2 promotes NSC clustering through N-cadherin expression. The insight gained about the mechanisms by which EC promote NSC migration may enhance NSC therapeutic response to sites of injury.

ECM hydrogel improves the delivery of PEG microsphere-encapsulated neural stem cells and endothelial cells into tissue cavities caused by stroke.

Intracerebral implantation of neural stem cells (NSCs) to treat stroke remains an inefficient process with <5% of injected cells being retained. To improve the retention and distribution of NSCs after a stroke, we investigated the utility of NSCs' encapsulation in polyethylene glycol (PEG) microspheres. We first characterized the impact of the physical properties of different syringes and needles, as well as ejection speed, upon delivery of microspheres to the stroke injured rat brain. A 20 G needle size at a 10 µL/min flow rate achieved the most efficient microsphere ejection. Secondly, we optimized the delivery vehicles for in vivo implantation of PEG microspheres. The suspension of microspheres in extracellular matrix (ECM) hydrogel showed superior retention and distribution in a cortical stroke caused by photothrombosis, as well as in a striatal and cortical cavity ensuing middle cerebral artery occlusion (MCAo). Thirdly, NSCs or NSCs + endothelial cells (ECs) encapsulated into biodegradable microspheres were implanted into a large stroke cavity. Cells in microspheres exhibited a high viability, survived freezing and transport. Implantation of 110 cells/microsphere suspended in ECM hydrogel produced a highly efficient delivery that resulted in the widespread distribution of NSCs in the tissue cavity and damaged peri-infarct tissues. Co-delivery of ECs enhanced the in vivo survival and distribution of ∼1.1 million NSCs. The delivery of NSCs and ECs can be dramatically improved using microsphere encapsulation combined with suspension in ECM hydrogel. These biomaterial innovations are essential to advance clinical efforts to improve the treatment of stroke using intracerebral cell therapy.

Author Correction: Minimally Invasive Delivery of Microbeads with Encapsulated, Viable and Quiescent Neural Stem Cells to the Adult Subventricular Zone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Minimally Invasive Delivery of Microbeads with Encapsulated, Viable and Quiescent Neural Stem Cells to the Adult Subventricular Zone.

Stem cell therapies demonstrate promising results as treatment for neurological disease and injury, owing to their innate ability to enhance endogenous neural tissue repair and promote functional recovery. However, delivery of undifferentiated and viable neuronal stem cells requires an engineered delivery system that promotes integration of transplanted cells into the inflamed and cytotoxic region of damaged tissue. Within the brain, endothelial cells (EC) of the subventricular zone play a critical role in neural stem cell (NSC) maintenance, quiescence and survival. Therefore, here, we describe the use of polyethylene glycol microbeads for the coincident delivery of EC and NSC as a means of enhancing appropriate NSC quiescence and survival during transplantation into the mouse brain. We demonstrate that EC and NSC co-encapsulation maintained NSC quiescence, enhanced NSC viability, and facilitated NSC extravasation in vitro, as compared to NSC encapsulated alone. In addition, co-encapsulated cells delivered to an in vivo non-injury model reduced inflammatory response compared to freely injected NSC. These results suggest the strong potential of a biomimetic engineered niche for NSC delivery into the brain following neurological injury.

Engineered Biomimetic Neural Stem Cell Niche.

PURPOSE OF REVIEW: Neural stem cells (NSCs) have the potential to proliferate and differentiate into functional neurons, heightening their potential use for therapeutic applications. This review explores bioengineered systems which recapitulate NSC niche cell-cell and cell-matrix interactions. RECENT FINDINGS: Delivery of NSCs to the cytotoxic injured brain is limited by low cell survival rates post-transplantation and poor maintenance of native niche bioactive components. The use of biomaterial platforms can mimic in vivo the environment of the two germinal areas of the adult brain in which NSCs thrive. An environmental mimic that includes extracellular proteins and moieties, along with appropriate biomechanical cues has recently demonstrated promising results in enhancing neurogenesis, aiding the production of a bioengineered niche. SUMMARY: Biocomposition, biomechanics, and biostructure can be manipulated through engineered platforms to re-create the biofunctionality of an NSC niche. Upon transplantation and delivery with biomimetic scaffolds, NSCs show potential to promote functional recovery and rebuild neural circuitry post neurological trauma.

Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disorders. Pericytes (PCs) have been implicated in the regulation of neutrophil transmigration, and previous work demonstrates that endothelial cell (EC)-derived signals reduce PC barrier function; however, the signaling mechanisms are unknown. Here, we demonstrate a novel role for EC-derived macrophage migration inhibitory factor (MIF) in inhibiting PC contractility and facilitating neutrophil transmigration. With the use of micro-ELISAs, RNA sequencing, quantitative PCR, and flow cytometry, we found that ECs secrete MIF, and PCs upregulate CD74 in response to TNF-α. We demonstrate that EC-derived MIF decreases PC contractility on 2-dimensional silicone substrates via reduction of phosphorylated myosin light chain. With the use of an in vitro microvascular model of the human EC-PC barrier, we demonstrate that MIF decreases the PC barrier to human neutrophil transmigration by increasing intercellular PC gap formation. For the first time, an EC-specific MIF knockout mouse was used to investigate the effects of selective deletion of EC MIF. In a model of acute lung injury, selective deletion of EC MIF decreases neutrophil infiltration to the bronchoalveolar lavage and tissue and simultaneously decreases PC relaxation by increasing myosin light-chain phosphorylation. We conclude that paracrine signals from EC via MIF decrease PC contraction and enhance PC-regulated neutrophil transmigration.-Pellowe, A. S., Sauler, M., Hou, Y., Merola, J., Liu, R., Calderon, B., Lauridsen, H. M., Harris, M. R., Leng, L., Zhang, Y., Tilstam, P. V., Pober, J. S., Bucala, R., Lee, P. J., Gonzalez, A. L. Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA-expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung-conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-ß1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.