Intracellular pH dynamics respond to microenvironment stiffening and mediate vasculogenic mimicry through ß-catenin.

Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we use two metastatic cell models, one breast and one lung, to assess pHi response to varying extracellular matrix (ECM) stiffness. To experimentally model ECM stiffening, we use two tunable-stiffness hydrogel systems: Matrigel and hyaluronic acid (HA) gels, which mimic the increased protein secretion and crosslinking associated with ECM stiffening. We find that single-cell pHi decreases with increased ECM stiffness in both hydrogel systems and both metastatic cell types. We also observed that stiff ECM promotes vasculogenic mimicry (VM), a phenotype associated with metastasis and resistance. Importantly, we show that decreased pHi is both a necessary and sufficient mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes and lowering pHi on soft ECM drives VM. We characterize ß-catenin as a pH-dependent molecular mediator of pH-dependent VM, where stiffness-driven changes in ß-catenin abundance can be overridden by increased pHi. We uncover a dynamic relationship between matrix stiffness and pHi, thus suggesting pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment.

Mimicking blood and lymphatic vasculatures using microfluidic systems.

The role of the circulatory system, containing the blood and lymphatic vasculatures, within the body, has become increasingly focused on by researchers as dysfunction of either of the systems has been linked to serious complications and disease. Currently, in vivo models are unable to provide the sufficient monitoring and level of manipulation needed to characterize the fluidic dynamics of the microcirculation in blood and lymphatic vessels; thus in vitro models have been pursued as an alternative model. Microfluidic devices have the required properties to provide a physiologically relevant circulatory system model for research as well as the experimental tools to conduct more advanced research analyses of microcirculation flow. In this review paper, the physiological behavior of fluid flow and electrical communication within the endothelial cells of the systems are detailed and discussed to highlight their complexities. Cell co-culturing methods and other relevant organ-on-a-chip devices will be evaluated to demonstrate the feasibility and relevance of the in vitro microfluidic model. Microfluidic systems will be determined as a noteworthy model that can display physiologically relevant flow of the cardiovascular and lymphatic systems, which will enable researchers to investigate the systems' prevalence in diseases and identify potential therapeutics.

The Effects of Preeclamptic Milieu on Cord Blood Derived Endothelial Colony-Forming Cells.

Preeclampsia is one of the leading causes of infant and maternal mortality worldwide. Many infants born from preeclamptic pregnancies are born prematurely with higher risk of developing cardiovascular later in their life. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). To gain insight into this, cord blood derived ECFCs isolated from preeclamptic pregnancies (PRECs) were analyzed and compared to their healthy counterparts. While PRECs preserve key endothelial markers, they upregulate several markers associated with oxidative stress and inflammatory response. Compared to ECFCs, PRECs also exhibit lower migratory behaviors and impaired angiogenic potential. Interestingly, treatment of neuropilin-1 can improve tube formation in vitro. Collectively, this study reports that preeclamptic milieu influence phenotypes and functionality of PRECs, which can be rejuvenated using exogenous molecules. Promising results from this study warrant future investigations on the prospect of the rejuvenated PRECs to improve lung function of infants born from preeclamptic pregnancies.

Synthetic hyaluronic acid coating preserves the phenotypes of lymphatic endothelial cells.

Lymphatic endothelial cells (LECs) play a critical role in the formation and maintenance of the lymphatic vasculature, which is essential for the immune system, fluid balance, and tissue repair. However, LECs are often difficult to study in vivo and in vitro models that accurately mimic their behaviors and phenotypes are limited. In particular, LECs have been shown to lose their lymphatic markers over time while being cultured in vitro, which reflect their plasticity and heterogeneity in vivo. Since LECs uniquely express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), we hypothesized that surface coating with hyaluronic acid (HA) can preserve LEC phenotypes and functionalities. Dopamine conjugated hyaluronic acid (HA-DP) was synthesized with 42% degree of substitution to enable surface modification and conjugation onto standard tissue culture plates. Compared to fibronectin coating and tissue culture plate controls, surface coating with HA-DP was able to preserve lymphatic markers, such as prospero homeobox protein 1 (Prox1), podoplanin (PDPN), and LYVE-1 over several passages in vitro. LECs cultured on HA-DP expressed lower levels of focal adhesion kinase (FAK) and YAP/TAZ, which may be responsible for the maintenance of the lymphatic characteristics. Collectively, the HA-DP coating may provide a novel method for culturing human LECs in vitro toward more representative studies in basic lymphatic biology and lymphatic regeneration.

Podoplanin is Responsible for the Distinct Blood and Lymphatic Capillaries.

Introduction: Controlling the formation of blood and lymphatic vasculatures is crucial for engineered tissues. Although the lymphatic vessels originate from embryonic blood vessels, the two retain functional and physiological differences even as they develop in the vicinity of each other. This suggests that there is a previously unknown molecular mechanism by which blood (BECs) and lymphatic endothelial cells (LECs) recognize each other and coordinate to generate distinct capillary networks. Methods: We utilized Matrigel and fibrin assays to determine how cord-like structures (CLS) can be controlled by altering LEC and BEC identity through podoplanin (PDPN) and folliculin (FLCN) expressions. We generated BEC ΔFLCN and LEC ΔPDPN , and observed cell migration to characterize loss lymphatic and blood characteristics due to respective knockouts. Results: We observed that LECs and BECs form distinct CLS in Matrigel and fibrin gels despite being cultured in close proximity with each other. We confirmed that the LECs and BECs do not recognize each other through paracrine signaling, as proliferation and migration of both cells were unaffected by paracrine signals. On the other hand, we found PDPN to be the key surface protein that is responsible for LEC-BEC recognition, and LECs lacking PDPN became pseudo-BECs and vice versa. We also found that FLCN maintains BEC identity through downregulation of PDPN. Conclusions: Overall, these observations reveal a new molecular pathway through which LECs and BECs form distinct CLS through physical contact by PDPN which in turn is regulated by FLCN, which has important implications toward designing functional engineered tissues. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00730-2.

Engineering bioactive nanoparticles to rejuvenate vascular progenitor cells.

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including type-2 diabetes mellitus, hypertension, and cardiovascular disease. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). Although several approaches have been previously explored to restore endothelial function, their widespread adoption remains tampered by systemic side effects of adjuvant drugs and unintended immune response of gene therapies. Here, we report a strategy to rejuvenate circulating vascular progenitor cells by conjugation of drug-loaded liposomal nanoparticles directly to the surface of GDM-exposed ECFCs (GDM-ECFCs). Bioactive nanoparticles can be robustly conjugated to the surface of ECFCs without altering cell viability and key progenitor phenotypes. Moreover, controlled delivery of therapeutic drugs to GDM-ECFCs is able to normalize transgelin (TAGLN) expression and improve cell migration, which is a critical key step in establishing functional vascular networks. More importantly, sustained pseudo-autocrine stimulation with bioactive nanoparticles is able to improve in vitro and in vivo vasculogenesis of GDM-ECFCs. Collectively, these findings highlight a simple, yet promising strategy to rejuvenate GDM-ECFCs and improve their therapeutic potential. Promising results from this study warrant future investigations on the prospect of the proposed strategy to improve dysfunctional vascular progenitor cells in the context of other chronic diseases, which has broad implications for addressing various cardiovascular complications, as well as advancing tissue repair and regenerative medicine.

Harnessing biomaterials for lymphatic system modulation.

The lymphatic system plays an integral part in regulating immune cell trafficking and the transport of macromolecules. However, its influence on disease progression and drug uptake is understood less than that of the vascular system. To bridge this knowledge gap, biomaterials can be used to investigate the lymphatic system and to provide novel understanding into complex disease processes, including cancer metastasis and inflammation. Insight gained from these mechanistic studies can be further used to design innovative biomaterials to modulate the immune system, improve drug delivery, and promote tissue regeneration. This review article focuses on recent advances in (i) biomaterials used for lymphatic vessel formation, (ii) models for studying lymphatic-immune cells interactions, (iii) pharmaceuticals and their interactions with the lymphatic system, (iv) and strategies for drug delivery via the lymphatic system. Finally, several challenges regarding adopting biomaterials for immunomodulation and future perspectives are discussed. STATEMENT OF SIGNIFICANCE: The lymphatic system plays an integral part in regulating immune cell trafficking and the transport of macromolecules. However, its influence on disease progression and drug uptake is understood less than that of the vascular system. This review article focuses on recent progresses in biomaterials to investigate the lymphatic system and to provide novel understanding into complex disease states. Insight gained from these mechanistic studies can be further used to design innovative biomaterials to modulate the immune system, improve drug delivery, and promote tissue regeneration. Finally, a number of challenges in adopting biomaterials for immunomodulation and future perspectives are discussed.