Dysfunctional mechanotransduction regulates the progression of PIK3CA-driven vascular malformations.

Somatic activating mutations in PIK3CA are common drivers of vascular and lymphatic malformations. Despite common biophysical signatures of tissues susceptible to lesion formation, including compliant extracellular matrix and low rates of perfusion, lesions vary in clinical presentation from localized cystic dilatation to diffuse and infiltrative vascular dysplasia. The mechanisms driving the differences in disease severity and variability in clinical presentation and the role of the biophysical microenvironment in potentiating progression are poorly understood. Here, we investigate the role of hemodynamic forces and the biophysical microenvironment in the pathophysiology of vascular malformations, and we identify hemodynamic shear stress and defective endothelial cell mechanotransduction as key regulators of lesion progression. We found that constitutive PI3K activation impaired flow-mediated endothelial cell alignment and barrier function. We show that defective shear stress sensing in PIK3CA E542K endothelial cells is associated with reduced myosin light chain phosphorylation, junctional instability, and defective recruitment of vinculin to cell-cell junctions. Using 3D microfluidic models of the vasculature, we demonstrate that PIK3CA E542K microvessels apply reduced traction forces and are unaffected by flow interruption. We further found that draining transmural flow resulted in increased sprouting and invasion responses in PIK3CA E542K microvessels. Mechanistically, constitutive PI3K activation decreased cellular and nuclear elasticity resulting in defective cellular tensional homeostasis in endothelial cells which may underlie vascular dilation, tissue hyperplasia, and hypersprouting in PIK3CA-driven venous and lymphatic malformations. Together, these results suggest that defective nuclear mechanics, impaired cellular mechanotransduction, and maladaptive hemodynamic responses contribute to the development and progression of PIK3CA-driven vascular malformations.

Human Cell-Derived Matrix Composite Hydrogels with Diverse Composition for Use in Vasculature-on-chip Models.

Microphysiological and organ-on-chip platforms seek to address critical gaps in human disease models and drug development that underlie poor rates of clinical success for novel interventions. While the fabrication technology and model cells used to synthesize organs-on-chip have advanced considerably, most platforms rely on animal-derived or synthetic extracellular matrix as a cell substrate, limiting mimicry of human physiology and precluding use in modeling diseases in which matrix dynamics play a role in pathogenesis. Here, the development of human cell-derived matrix (hCDM) composite hydrogels for use in 3D microphysiologic models of the vasculature is reported. hCDM composite hydrogels are derived from human donor fibroblasts and maintain a complex milieu of basement membrane, proteoglycans, and nonfibrillar matrix components. The use of hCDM composite hydrogels as 2D and 3D cell culture substrates is demonstrated, and hCDM composite hydrogels are patterned to form engineered human microvessels. Interestingly, hCDM composite hydrogels are enriched in proteins associated with vascular morphogenesis as determined by mass spectrometry, and functional analysis demonstrates proangiogenic signatures in human endothelial cells cultured in these hydrogels. In conclusion, this study suggests that human donor-derived hCDM composite hydrogels could address technical gaps in human organs-on-chip development and serve as substrates to promote vascularization.

Vascular organs-on-chip made with patient-derived endothelial cells: technologies to transform drug discovery and disease modeling.

INTRODUCTION: Vascular diseases impart a tremendous burden on healthcare systems in the United States and across the world. Efforts to improve therapeutic interventions are hindered by limitations of current experimental models. The integration of patient-derived cells with organ-on-chip (OoC) technology is a promising avenue for preclinical drug screening that improves upon traditional cell culture and animal models. AREAS COVERED: The authors review induced pluripotent stem cells (iPSC) and blood outgrowth endothelial cells (BOEC) as two sources for patient-derived endothelial cells (EC). They summarize several studies that leverage patient-derived EC and OoC for precision disease modeling of the vasculature, with a focus on applications for drug discovery. They also highlight the utility of patient-derived EC in other translational endeavors, including ex vivo organogenesis and multi-organ-chip integration. EXPERT OPINION: Precision disease modeling continues to mature in the academic space, but end-use by pharmaceutical companies is currently limited. To fully realize their transformative potential, OoC systems must balance their complexity with their ability to integrate with the highly standardized and high-throughput experimentation required for drug discovery and development.

Aminobisphosphonates reactivate the latent reservoir in people living with HIV-1.

Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy. Current research efforts to cure HIV-1 infection include "shock and kill" strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells. The modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy. Aminobisphosphonates (N-BPs) that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies. Here, we show the use of N-BPs as a novel class of LRA: we found in ex vivo assays using primary cells from ART-suppressed people living with HIV-1 that N-BPs induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin (PHA). RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further evidence of alendronate-mediated latency reversal and activation of immune effector cells. Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of N-BPs to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination.

Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics.

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. STATEMENT OF SIGNIFICANCE: The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.

Aminobisphosphonates reactivate the latent reservoir in people living with HIV-1.

Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy. Current research efforts to cure HIV-1 infection include "shock and kill" strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells. The modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy. Aminobisphosphonates (N-BPs) that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies. Here, we show the use of N-BPs as a novel class of LRA: we found in ex vivo assays using primary cells from ART-suppressed people living with HIV-1 that N-BPs induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin (PHA). RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further evidence of alendronate-mediated latency reversal and activation of immune effector cells. Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of N-BPs to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination.

Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation.

Somatic activating mutations of PIK3CA are associated with development of vascular malformations (VMs). Here, we describe a microfluidic model of PIK3CA-driven VMs consisting of human umbilical vein endothelial cells expressing PIK3CA activating mutations embedded in three-dimensional hydrogels. We observed enlarged, irregular vessel phenotypes and the formation of cyst-like structures consistent with clinical signatures and not previously observed in cell culture models. Pathologic morphologies occurred concomitant with up-regulation of Rac1/p21-activated kinase (PAK), mitogen-activated protein kinase cascades (MEK/ERK), and mammalian target of rapamycin (mTORC1/2) signaling networks. We observed differential effects between alpelisib, a PIK3CA inhibitor, and rapamycin, an mTORC1 inhibitor, in mitigating matrix degradation and network topology. While both were effective in preventing vessel enlargement, rapamycin failed to reduce MEK/ERK and mTORC2 activity and resulted in hyperbranching, while inhibiting PAK, MEK1/2, and mTORC1/2 mitigates abnormal growth and vascular dilation. Collectively, these findings demonstrate an in vitro platform for VMs and establish a role of dysregulated Rac1/PAK and mTORC1/2 signaling in PIK3CA-driven VMs.

γδ T cells: an immunotherapeutic approach for HIV cure strategies.

Current strategies aimed to cure HIV infection are based on combined efforts to reactivate the virus from latency and improve immune effector cell function to clear infected cells. These strategies are primarily focused on CD8+ T cells and approaches are challenging due to insufficient HIV antigen production from infected cells and poor HIV-specific CD8+ T cells. γδ T cells represent a unique subset of effector T cells that can traffic to tissues, and selectively target cancer or virally infected cells without requiring MHC presentation. We analyzed whether γδ T cells represent a complementary/alternative immunotherapeutic approach towards HIV cure strategies. γδ T cells from HIV-infected virologically suppressed donors were expanded with bisphosphonate pamidronate (PAM) and cells were used in autologous cellular systems ex vivo. These cells (a) are potent cytotoxic effectors able to efficiently inhibit HIV replication ex vivo, (b) degranulate in the presence of autologous infected CD4+ T cells, and (c) specifically clear latently infected cells after latency reversal with vorinostat. This is the first proof of concept to our knowledge showing that γδ T cells target and clear autologous HIV reservoirs upon latency reversal. Our results open potentially new insights into the immunotherapeutic use of γδ T cells for current interventions in HIV eradication strategies.