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Abstract A recently discovered but unexplored mechanism of angiogenesis regulation is cross-family binding between platelet-derived growth factors (PDGFs) and vascular endothelial growth factor receptors (VEGFRs), which suggests a novel therapy for addressing vascular dysregulation. This study elucidates the role of PDGFs in endothelial cell (EC) signaling and functions, focusing on VEGFR activation. Using human dermal microvascular ECs (HDMECs) with double knockout of PDGFRα/β and human brain microvascular ECs (HBMECs), we show three key findings: (1) PDGF-AA and -BB induced VEGFR1 phosphorylation, peaking at 2-fold increases at low concentrations (0.5 ng/mL), while PDGF-AB stimulated a 2-fold rise in VEGFR2 phosphorylation. (2) Downstream effectors PLCγ1, Akt, and FAK were activated by all three PDGFs at levels comparable to VEGF-A, achieving approximately 70% of VEGF-A’s effects. (3) PDGF-BB significantly enhanced EC proliferation (up to 240%) and migration (up to 170%), with lower PDGF concentrations (0.5–5 ng/mL) eliciting stronger effects than higher concentrations (50–100 ng/mL). Overall, PDGF subtypes differentially induce VEGFR phosphorylation, downstream effector activation, and angiogenic hallmarks such as proliferation and migration, revealing novel mechanisms for regulating endothelial function. 

Abstract PurposeReceptor tyrosine kinase (RTK) concentrations on the plasma membrane correlate with angiogenic functions in vitro and in rodent models. The intracellular RTK pool also regulates plasma membrane receptor availability and signaling pathways. Organs have specialized angiogenic functions essential to their distinct roles, supporting the hypothesis that plasma membrane and intracellular RTK concentrations vary across endothelial cells (ECs) from different organs. MethodsUsing quantitative flow cytometry on human ECs derived from dermis, umbilical vein, kidney, liver, and brain, we measured and statistically analyzed the concentrations of selected RTKs within ECs and on their plasma membranes. ResultsVEGFR1 exhibited the lowest concentrations on the plasma membrane (300–900 VEGFR1/cell) among VEGFRs. HDMECs (dermis) showed the lowest VEGFR1 level among the examined EC types. Whole-cell VEGFR1 concentrations were 2500–7500 VEGFR1/cell, with 12–26% located on the plasma membrane. The proportion of VEGFR2 located on the plasma membrane was higher at > 30%, except in HGMECs (kidney) where it was 24%. Plasma membrane VEGFR2 was significantly lower in HDMECs and HGMECs compared with HBMECs (brain), whereas whole-cell VEGFR2 levels were consistently in the range of 14,100–22,500 molecules/cell. VEGFR3 was the least localized to the plasma membrane, from 2% in HGMECs to 14% in HDMECs at the highest level of 4400 VEGFR3/cell. Whole-cell VEGFR3 concentrations ranged from 32,400 in HDMECs to 62,000 VEGFR3/cell in HLiSMECs (liver), with no significant differences among EC types. NRP1 was most abundant on the plasma membrane of HUVECs (umbilical vein) at 39,700 NRP1/cell; other ECs displayed 26,000–29,900 NRP1/cell, approximately 5-fold higher than the numbers of VEGFRs. Across EC types, Axl was present on the plasma membrane at levels (6900–12,200 Axl/cell) similar to those of VEGFR2. ConclusionsWe quantified and statistically analyzed plasma membrane and whole-cell expression of angiogenic RTKs across cultured human ECs from five different organs. Our findings suggest that RTK protein distribution might not fully reflect the differential angiogenic capacities in cultured ECs. In vitro monoculture conditions might reduce EC organ-specific features essential for refining vascular models. 

Abstract Angiogenesis, the formation of new vessels from existing vessels, is mediated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). Despite discoveries supporting the cross-family interactions between VEGF and PDGF families, sharing the binding partners between them makes it challenging to identify growth factors that predominantly affect angiogenesis. Systems biology offers promises to untangle this complexity. Thus, in this study, we developed a mass-action kinetics-based computational model for cross-family interactions between VEGFs (VEGF-A, VEGF-B, and PlGF) and PDGFs (PDGF-AA, PDGF-AB, and PDGF-BB) with their receptors (VEGFR1, VEGFR2, NRP1, PDGFRα, and PDGFRβ). The model, parametrized with our literature mining and surface resonance plasmon assays, was validated by comparing the concentration of VEGFR1 complexes with a previously constructed angiogenesis model. The model predictions include five outcomes: 1) the percentage of free or bound ligands and 2) receptors, 3) the concentration of free ligands, 4) the percentage of ligands occupying each receptor, and 5) the concentration of ligands that is bound to each receptor. We found that at equimolar ligand concentrations (1 nM), PlGF and VEGF-A were the main binding partners of VEGFR1 and VEGFR2, respectively. Varying the density of receptors resulted in the following five outcomes: 1) Increasing VEGFR1 density depletes the free PlGF concentration, 2) increasing VEGFR2 density decreases PDGF:PDGFRα complexes, 3) increased NRP1 density generates a biphasic concentration of the free PlGF, 4) increased PDGFRα density increases PDGFs:PDGFRα binding, and 5) increasing PDGFRβ density increases VEGF-A:PDGFRβ. Our model offers a reproducible, fundamental framework for exploring cross-family interactions that can be extended to the tissue level or intracellular molecular level. Also, our model may help develop therapeutic strategies in pathological angiogenesis by identifying the dominant complex in the cell signaling. Author summaryNew blood vessel formation from existing ones is essential for growth, healing, and reproduction. However, when this process is disrupted—either too much or too little—it can contribute to diseases such as cancer and peripheral arterial disease. Two key families of proteins, vascular endothelial growth factors (VEGFs) and platelet-derived growth factors (PDGFs), regulate this process. Traditionally, scientists believed that VEGFs only bind to VEGF receptors and PDGFs to PDGF receptors. However, recent findings show that these proteins can interact with each other’s receptors, making it more challenging to understand and control blood vessel formation. To clarify these complex interactions, we combined computer modeling with biological data to map out which proteins bind to which receptors and to what extent. Our findings show that when VEGFs and PDGFs are present in equal amounts, VEGFs are the primary binding partners for VEGF receptors. We also explored how changes in receptor levels affect these interactions in disease-like conditions. This work provides a foundational computational model for studying cross-family interactions, which can be expanded to investigate tissue-level effects and processes inside cells. Ultimately, our model may help develop better treatments for diseases linked to abnormal blood vessel growth by identifying key protein-receptor interactions. 

ABSTRACT Inadequate angiogenesis in obesogenic adipose tissue (AT) has been implicated in disrupted adipogenesis and metabolic disorders. Yet, key cellular and molecular regulators of AT angiogenesis remain largely unidentified. This study sought to identify the dysregulated elements within the Vascular Endothelial Growth Factor (VEGF) and Platelet-Derived Growth Factor (PDGF) systems during obesity progression. We employ a mouse model, comprising both male and female mice, to investigate the changes in the VEGF/PDGF concentration and their receptor distribution in AT during short- and long-term weight gain and weight loss. Our results reveal pronounced sex-specific differences in obesity progression, with male and female mice exhibiting distinct angiogenic ligand and receptor profiles under identical dietary interventions. This data also lays the groundwork for developing computational models of VEGF/PDGF signaling networks in AT, allowing for the simulation of complex biological interactions and the prediction of therapeutic outcomes. 

Abstract Obesity is a global health crisis, with its prevalence particularly severe in the United States, where over 42% of adults are classified as obese. Obesity is driven by complex molecular and tissue-level mechanisms that remain poorly understood. Among these, angiogenesis—primarily mediated by vascular endothelial growth factor (VEGF-A)—is critical for adipose tissue expansion but presents unique challenges for therapeutic targeting due to its intricate regulation. Systems biology approaches have advanced our understanding of VEGF-A signaling in vascular diseases, but their application to obesity is limited by scattered and sometimes contradictory data. To address this gap, we performed a comprehensive analysis of the existing literature to synthesize key findings, standardize data, and provide a holistic perspective on the adipose vascular microenvironment. The data mining revealed five key findings: (1) obesity increases adipocyte size by 78%; (2) vessel density in adipose tissue decreases by 51% in obese mice, with vessels being 47–58% smaller and 4–9 times denser in comparison with tumor vessels; (3) capillary basement membrane thickness remains similar regardless of obesity; (4) VEGF-A shows the strongest binding affinity for VEGFR1, with four times stronger affinity for VEGFR2 than for NRP1; and (5) binding affinities measured by radioligand binding assay and surface plasmon resonance (SPR) are significantly different. These consolidated findings provide essential parameters for systems biology modeling, new insights into obesity-induced changes in adipose tissue, and a foundation for developing angiogenesis-targeting therapies for obesity. 

Oxytocin acts through the oxytocin receptor (OXTR) to modulate uterine contractility. We previously identified OXTR genetic variants and showed that, in HEK293T cells, two of the OXTR protein variants localized to the cell surface less than wild-type OXTR. Here, we sought to measure OXTR in the more native human myometrial smooth muscle cell (HMSMC) line on both the cell-surface and across the whole cell, and used CRISPR editing to add an HA tag to the endogenous OXTR gene for anti-HA measurement. Quantitative flow cytometry revealed that these cells possessed 55,000 ± 3200 total OXTRs and 4900 ± 390 cell-surface OXTRs per cell. To identify any differential wild-type versus variant localization, we transiently transfected HMSMCs to exogenously express wild-type or variant OXTR with HA and green fluorescent protein tags. Total protein expression of wild-type OXTR and all tested variants were similar. However, the two variants with lower surface localization in HEK293T cells also presented lower surface localization in HMSMCs. Overall, we confirm the differential surface localization of variant OXTR in a more native cell type, and further demonstrate that the quantitative flow cytometry technique is adaptable to whole-cell measurements. 

Abstract IntroductionAbnormal angiogenesis is central to vascular disease and cancer, and noninvasive biomarkers of vascular origin are needed to evaluate patients and therapies. Vascular endothelial growth factor receptors (VEGFRs) are often dysregulated in these diseases, making them promising biomarkers, but the need for an invasive biopsy has limited biomarker research on VEGFRs. Here, we pioneer a blood biopsy approach to quantify VEGFR plasma membrane localization on two circulating vascular proxies: circulating endothelial cells (cECs) and circulating progenitor cells (cPCs). MethodsUsing quantitative flow cytometry, we examined VEGFR expression on cECs and cPCs in four age-sex groups: peri/premenopausal females (aged < 50 years), menopausal/postmenopausal females (≥ 50 years), and younger and older males with the same age cut-off (50 years). ResultscECs in peri/premenopausal females consisted of two VEGFR populations: VEGFR-low (~ 55% of population: population medians ~ 3000 VEGFR1 and 3000 VEGFR2/cell) and VEGFR-high (~ 45%: 138,000 VEGFR1 and 39,000–236,000 VEGFR2/cell), while the menopausal/postmenopausal group only possessed the VEGFR-low cEC population; and 27% of cECs in males exhibited high plasma membrane VEGFR expression (206,000 VEGFR1 and 155,000 VEGFR2/cell). The absence of VEGFR-high cEC subpopulations in menopausal/postmenopausal females suggests that their high-VEGFR cECs are associated with menstruation and could be noninvasive proxies for studying the intersection of age-sex in angiogenesis. VEGFR1 plasma membrane localization in cPCs was detected only in menopausal/postmenopausal females, suggesting a menopause-specific regenerative mechanism. ConclusionsOverall, our quantitative, noninvasive approach targeting cECs and cPCs has provided the first insights into how sex and age influence VEGFR plasma membrane localization in vascular cells.