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.

Optimal Performance Objectives in the Highly Conserved Bone Morphogenetic Protein Signaling Pathway.

Throughout development, complex networks of cell signaling pathways drive cellular decision-making across different tissues and contexts. The transforming growth factor ß (TGF-ß) pathways, including the BMP/Smad pathway, play crucial roles in these cellular responses. However, as the Smad pathway is used reiteratively throughout the life cycle of all animals, its systems-level behavior varies from one context to another, despite the pathway connectivity remaining nearly constant. For instance, some cellular systems require a rapid response, while others require high noise filtering. In this paper, we examine how the BMP- Smad pathway balances trade-offs among three such systems-level behaviors, or "Performance Objectives (POs)": response speed, noise amplification, and the sensitivity of pathway output to receptor input. Using a Smad pathway model fit to human cell data, we show that varying non-conserved parameters (NCPs) such as protein concentrations, the Smad pathway can be tuned to emphasize any of the three POs and that the concentration of nuclear phosphatase has the greatest effect on tuning the POs. However, due to competition among the POs, the pathway cannot simultaneously optimize all three, but at best must balance trade-offs among the POs. We applied the multi-objective optimization concept of the Pareto Front, a widely used concept in economics to identify optimal trade-offs among various requirements. We show that the BMP pathway efficiently balances competing POs across species and is largely Pareto optimal. Our findings reveal that varying the concentration of NCPs allows the Smad signaling pathway to generate a diverse range of POs. This insight identifies how signaling pathways can be optimally tuned for each context.

Hyaluronic Acid Hydrogels with Phototunable Supramolecular Cross-Linking for Spatially Controlled Lymphatic Tube Formation.

The dynamics of the extracellular matrix (ECM) influences stem cell differentiation and morphogenesis into complex lymphatic networks. While dynamic hydrogels with stress relaxation properties have been developed, many require detailed chemical processing to tune viscoelasticity, offering a limited opportunity for in situ and spatiotemporal control. Here, a hyaluronic acid (HA) hydrogel is reported with viscoelasticity that is controlled and spatially tunable using UV light to direct the extent of supramolecular and covalent cross-linking interactions. This is achieved using UV-mediated photodimerization of a supramolecular ternary complex of pendant trans-Brooker's Merocyanine (BM) guests and a cucurbit[8]uril (CB[8]) macrocycle. The UV-mediated conversion of this supramolecular complex to its covalent photodimerized form is catalyzed by CB[8], offering a user-directed route to spatially control hydrogel dynamics in combination with orthogonal photopatterning by UV irradiation through photomasks. This material thus achieves spatial heterogeneity of substrate dynamics, recreating features of native ECM without the need for additional chemical reagents. Moreover, these dynamic hydrogels afford spatial control of substrate mechanics to direct human lymphatic endothelial cells (LECs) to form lymphatic cord-like structures (CLS). Specifically, cells cultured on viscoelastic supramolecular hydrogels have enhanced formation of CLS, arising from increased expression of key lymphatic markers, such as LYVE-1, Podoplanin, and Prox1, compared to static elastic hydrogels prepared from fully covalent cross-linking. Viscoelastic hydrogels promote lymphatic CLS formation through the expression of Nrp2, VEGFR2, and VEGFR3 to enhance the VEGF-C stimulation. Overall, viscoelastic supramolecular hydrogels offer a facile route to spatially control lymphatic CLS formation, providing a tool for future studies of basic lymphatic biology and tissue engineering applications.

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.

Fractal dimension to characterize interactions between blood and lymphatic endothelial cells.

Spatial patterning of different cell types is crucial for tissue engineering and is characterized by the formation of sharp boundary between segregated groups of cells of different lineages. The cell-cell boundary layers, depending on the relative adhesion forces, can result in kinks in the border, similar to fingering patterns between two viscous partially miscible fluids which can be characterized by its fractal dimension. This suggests that mathematical models used to analyze the fingering patterns can be applied to cell migration data as a metric for intercellular adhesion forces. In this study, we develop a novel computational analysis method to characterize the interactions between blood endothelial cells (BECs) and lymphatic endothelial cells (LECs), which form segregated vasculature by recognizing each other through podoplanin. We observed indiscriminate mixing with LEC-LEC and BEC-BEC pairs and a sharp boundary between LEC-BEC pair, and fingering-like patterns with pseudo-LEC-BEC pairs. We found that the box counting method yields fractal dimension between 1 for sharp boundaries and 1.3 for indiscriminate mixing, and intermediate values for fingering-like boundaries. We further verify that these results are due to differential affinity by performing random walk simulations with differential attraction to nearby cells and generate similar migration pattern, confirming that higher differential attraction between different cell types result in lower fractal dimensions. We estimate the characteristic velocity and interfacial tension for our simulated and experimental data to show that the fractal dimension negatively correlates with capillary number (Ca), further indicating that the mathematical models used to study viscous fingering pattern can be used to characterize cell-cell mixing. Taken together, these results indicate that the fractal analysis of segregation boundaries can be used as a simple metric to estimate relative cell-cell adhesion forces between different cell types.

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.

Biomimetic Hydrogels to Promote Wound Healing.

Wound healing is a common physiological process which consists of a sequence of molecular and cellular events that occur following the onset of a tissue lesion in order to reconstitute barrier between body and external environment. The inherent properties of hydrogels allow the damaged tissue to heal by supporting a hydrated environment which has long been explored in wound management to aid in autolytic debridement. However, chronic non-healing wounds require added therapeutic features that can be achieved by incorporation of biomolecules and supporting cells to promote faster and better healing outcomes. In recent decades, numerous hydrogels have been developed and modified to match the time scale for distinct stages of wound healing. This review will discuss the effects of various types of hydrogels on wound pathophysiology, as well as the ideal characteristics of hydrogels for wound healing, crosslinking mechanism, fabrication techniques and design considerations of hydrogel engineering. Finally, several challenges related to adopting hydrogels to promote wound healing and future perspectives are discussed.

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.

Conformal single cell hydrogel coating with electrically induced tip streaming of an AC cone.

Encapsulation of single cells in a thin hydrogel provides a more precise control of stem cell niches and better molecular transport. Despite the recent advances in microfluidic technologies to allow encapsulation of single cells, existing methods rely on special crosslinking agents that are pre-coated on the cell surface and subject to the variation of the cell membrane, which limits their widespread adoption. This work reports a high-throughput single-cell encapsulation method based on the "tip streaming" mode of alternating current (AC) electrospray, with encapsulation efficiencies over 80% after tuned centrifugation. Dripping with multiple cells is curtailed due to gating by the sharp conic meniscus of the tip streaming mode that only allows one cell to be ejected at a time. Moreover, the method can be universally applied to both natural and synthetic hydrogels, as well as various cell types, including human multipotent mesenchymal stromal cells (hMSCs). Encapsulated hMSCs maintain good cell viability over an extended culture period and exhibit robust differentiation potential into osteoblasts and adipocytes. Collectively, electrically induced tip streaming enables high-throughput encapsulation of single cells with high efficiency and universality, which is applicable for various applications in cell therapy, pharmacokinetic studies, and regenerative medicine.

Matrix stiffness primes lymphatic tube formation directed by vascular endothelial growth factor-C.

Dysfunction of the lymphatic system is associated with a wide range of disease phenotypes. The restoration of dysfunctional lymphatic vessels has been hypothesized as an innovative method to rescue healthy phenotypes in diseased states including neurological conditions, metabolic syndromes, and cardiovascular disease. Compared to the vascular system, little is known about the molecular regulation that controls lymphatic tube morphogenesis. Using synthetic hyaluronic acid (HA) hydrogels as a chemically and mechanically tunable system to preserve lymphatic endothelial cell (LECs) phenotypes, we demonstrate that low matrix elasticity primes lymphatic cord-like structure (CLS) formation directed by a high concentration of vascular endothelial growth factor-C (VEGF-C). Decreasing the substrate stiffness results in the upregulation of key lymphatic markers, including PROX-1, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and VEGFR-3. Consequently, higher levels of VEGFR-3 enable stimulation of LECs with VEGF-C which is required to both activate matrix metalloproteinases (MMPs) and facilitate LEC migration. Both of these steps are critical in establishing CLS formation in vitro. With decreases in substrate elasticity, we observe increased MMP expression and increased cellular elongation, as well as formation of intracellular vacuoles, which can further merge into coalescent vacuoles. RNAi studies demonstrate that MMP-14 is required to enable CLS formation and that LECs sense matrix stiffness through YAP/TAZ mechanosensors leading to the activation of their downstream target genes. Collectively, we show that by tuning both the matrix stiffness and VEGF-C concentration, the signaling pathways of CLS formation can be regulated in a synthetic matrix, resulting in lymphatic networks which will be useful for the study of lymphatic biology and future approaches in tissue regeneration.

A multiplexed immuno-sensor for on-line and automated monitoring of tissue culture protein biomarkers.

Frequent on-line and automated monitoring of multiple protein biomarkers level secreted in the culture media during tissue growth is essential for the successful development of Tissue Engineering and Regenerative Medicine (TERM) products. Here, we present a low-cost, rapid, reliable, and integrable anion-exchange membrane-(AEM) based multiplexed sensing platform for this application. Unlike the gold-standard manual ELISA test, incubation/wash steps are optimized for each target and precisely metered in microfluidic chips to enhance selectivity. Unlike optical detection and unreliable visual detection for the ELISA test, which require standardization for every usage, the AEM ion current signal also offers robustness, endowed by the pH and ionic strength control capability of the ion-selective membrane, such that a universal standard curve can be used to calibrate all runs. The electrical signal is enhanced by highly charged silica nanoparticle reporters, which also act as hydrodynamic shear amplifiers to enhance selectivity during wash. This AEM-based sensing platform is tested with vascular protein biomarkers, Endothelin-1 (ET-1), Angiogenin (ANG) and Placental Growth Factor (PlGF). The limit of detection and three-decade dynamic range are comparable to ELISA assay but with a significantly reduced assay time of 1 h vs 7 h, due to the elimination of calibration and blocking steps. Optimized protocol for each target renders the detection highly reliable with more than 98% confidence. The multiplexed detection capability of the platform is also demonstrated by simultaneous detection of ET-1, ANG and PlGF in 40 µl of the vascular endothelial cell culture supernatants using three-membrane AEM sensor and the performance is validated against ELISA.

Membrane-Based Affinity Purification to Identify Target Proteins of a Small-Molecule Drug.

Identifying the target proteins of small-molecule drug candidates is important for determining their molecular mechanisms of action. Porous membranes derivatized with such small molecules may provide an attractive target-identification platform due to a high protein-capture efficiency during flow through membrane pores. This work employs carbonic anhydrase II (CAII) binding to immobilized 4-(2-aminoethyl)benzenesulfonamide (AEBSA) to examine the efficiency and selectivity of affinity capture in modified membranes. Selective elution of captured protein, tryptic digestion, tandem mass spectrometry analysis, and label-free quantification (LFQ) identify CAII as the dominant AEBSA target in diluted serum or cell lysate. CAII identification relies on determining the ratio of protein LFQ intensities in sample and control experiments, where free AEBSA added to the control loading solution limits CAII capture. Global proteomics shows that the spiked CAII is the only protein with a log2 ratio consistently >2, and the detection limit for CAII identification is 0.004 wt % of the total protein in 1:4 diluted human serum or 0.024 wt % of the total protein from breast cancer cell lysates. The same approach also identifies native CAII in human kidney cell lysate as an AEBSA target. Comparison of affinity capture using membranes, Affi-Gel 10 resin or M-270 Dynabeads derivatized with AEBSA suggests that only membranes allow identification of low-abundance CAII as a target.

Lymphatic Tissue Engineering and Regeneration.

The lymphatic system is a major circulatory system within the body, responsible for the transport of interstitial fluid, waste products, immune cells, and proteins. Compared to other physiological systems, the molecular mechanisms and underlying disease pathology largely remain to be understood which has hindered advancements in therapeutic options for lymphatic disorders. Dysfunction of the lymphatic system is associated with a wide range of disease phenotypes and has also been speculated as a route to rescue healthy phenotypes in areas including cardiovascular disease, metabolic syndrome, and neurological conditions. This review will discuss lymphatic system functions and structure, cell sources for regenerating lymphatic vessels, current approaches for engineering lymphatic vessels, and specific therapeutic areas that would benefit from advances in lymphatic tissue engineering and regeneration.

Platelet-targeted dual pathway antithrombotic inhibits thrombosis with preserved hemostasis.

Despite advances in antithrombotic therapy, the risk of recurrent coronary/cerebrovascular ischemia or venous thromboembolism remains high. Dual pathway antithrombotic blockade, using both antiplatelet and anticoagulant therapy, offers the promise of improved thrombotic protection; however, widespread adoption remains tempered by substantial risk of major bleeding. Here, we report a dual pathway therapeutic capable of site-specific targeting to activated platelets and therapeutic enrichment at the site of thrombus growth to allow reduced dosing without compromised antithrombotic efficacy. We engineered a recombinant fusion protein, SCE5-TAP, which consists of a single-chain antibody (SCE5) that targets and blocks the activated GPIIb/IIIa complex, and tick anticoagulant peptide (TAP), a potent direct inhibitor of activated factor X (FXa). SCE5-TAP demonstrated selective platelet targeting and inhibition of thrombosis in murine models of both carotid artery and inferior vena cava thrombosis, without a significant impact on hemostasis. Selective targeting to activated platelets provides an attractive strategy to achieve high antithrombotic efficacy with reduced risk of bleeding complications.

Rapid homogeneous endothelialization of high aspect ratio microvascular networks.

Microvascularization of an engineered tissue construct is necessary to ensure the nourishment and viability of the hosted cells. Microvascular constructs can be created by seeding the luminal surfaces of microfluidic channel arrays with endothelial cells. However, in a conventional flow-based system, the uniformity of endothelialization of such an engineered microvascular network is constrained by mass transfer of the cells through high length-to-diameter (L/D) aspect ratio microchannels. Moreover, given the inherent limitations of the initial seeding process to generate a uniform cell coating, the large surface-area-to-volume ratio of microfluidic systems demands long culture periods for the formation of confluent cellular microconduits. In this report, we describe the design of polydimethylsiloxane (PDMS) and poly(glycerol sebacate) (PGS) microvascular constructs with reentrant microchannels that facilitates rapid, spatially homogeneous endothelial cell seeding of a high L/D (2 cm/35 µm; > 550:1) aspect ratio microchannels. MEMS technology was employed for the fabrication of a monolithic, elastomeric, reentrant microvascular construct. Isotropic etching and PDMS micromolding yielded a near-cylindrical microvascular channel array. A 'stretch - seed - seal' operation was implemented for uniform incorporation of endothelial cells along the entire microvascular area of the construct yielding endothelialized microvascular networks in less than 24 h. The feasibility of this endothelialization strategy and the uniformity of cellularization were established using confocal microscope imaging.

Glycopeptide analogues of PSGL-1 inhibit P-selectin in vitro and in vivo.

Blockade of P-selectin (P-sel)/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogues. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-sel with nanomolar affinity (Kd~22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-sel/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.

Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix.

The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature's inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.