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1. Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity

   https://doi.org/10.3390/pr7060356  

   Chen, Si; Imoukhuede, P. I. (June 2019, Processes)

   Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for both normal development and numerous pathologies. Systems biology has offered a unique approach to study angiogenesis by profiling tyrosine kinase receptors (RTKs) that regulate angiogenic processes and computationally modeling RTK signaling pathways. Historically, this systems biology approach has been applied on ex vivo angiogenesis assays, however, these assays are difficult to quantify and limited in their potential of temporal analysis. In this study, we adopted a simple two-dimensional angiogenesis assay comprised of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) and examined temporal dynamics of a panel of six RTKs and cell heterogeneity up to 17 days. We observed ~2700 VEGFR1 (vascular endothelial growth factor receptor 1) per cell on 24-h-old cocultured HDF plasma membranes, which do not express VEGFR when cultured alone. We observed 4000–8100 VEGFR2 per cell on cocultured HUVEC plasma membranes throughout endothelial tube formation. We showed steady increase of platelet-derived growth factor receptors (PDGFRs) on cocultured HDF plasma membranes, and more interestingly, 1900–2900 PDGFRβ per plasma membrane were found on HUVECs within the first six hours of coculturing. These quantitative findings will offer us insights into molecular regulation during angiogenesis and help assess in vitro tube formation models and their physiological relevance. 

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2. Toward Blood-Based Precision Medicine: Identifying Age-Sex-Specific Vascular Biomarker Quantities on Circulating Vascular Cells

   https://doi.org/10.1007/s12195-023-00771-1  

   Fang, Yingye; Chen, Ling; Imoukhuede, P. I. (July 2023, Cellular and Molecular Bioengineering)

   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. 

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3. Cross-family Signaling: PDGF Mediates VEGFR Activation and Endothelial Function

   https://doi.org/10.1101/2025.02.27.640684  

   Liu, Xinming; Fang, Yingye; Imoukhuede, PI (March 2025, bioRxiv)

   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. 

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4. Integrative meta-modeling identifies endocytic vesicles, late endosome and the nucleus as the cellular compartments primarily directing RTK signaling

   https://doi.org/10.1039/C7IB00011A  

   Weddell, Jared C.; Imoukhuede, Princess I. (January 2017, Integrative Biology)

   Recently, intracellular receptor signaling has been identified as a key component mediating cell responses for various receptor tyrosine kinases (RTKs). However, the extent each endocytic compartment (endocytic vesicle, early endosome, recycling endosome, late endosome, lysosome and nucleus) contributes to receptor signaling has not been quantified. Furthermore, our understanding of endocytosis and receptor signaling is complicated by cell- or receptor-specific endocytosis mechanisms. Therefore, towards understanding the differential endocytic compartment signaling roles, and identifying how to achieve signal transduction control for RTKs, we delineate how endocytosis regulates RTK signaling. We achieve this via a meta-analysis across eight RTKs, integrating computational modeling with experimentally derived cell (compartment volume, trafficking kinetics and pH) and ligand–receptor (ligand/receptor concentration and interaction kinetics) physiology. Our simulations predict the abundance of signaling from eight RTKs, identifying the following hierarchy in RTK signaling: PDGFRβ > IGFR1 > EGFR > PDGFRα > VEGFR1 > VEGFR2 > Tie2 > FGFR1. We find that endocytic vesicles are the primary cell signaling compartment; over 43% of total receptor signaling occurs within the endocytic vesicle compartment for these eight RTKs. Mechanistically, we found that high RTK signaling within endocytic vesicles may be attributed to their low volume (5.3 × 10 −19 L) which facilitates an enriched ligand concentration (3.2 μM per ligand molecule within the endocytic vesicle). Under the analyzed physiological conditions, we identified extracellular ligand concentration as the most sensitive parameter to change; hence the most significant one to modify when regulating absolute compartment signaling. We also found that the late endosome and nucleus compartments are important contributors to receptor signaling, where 26% and 18%, respectively, of average receptor signaling occurs across the eight RTKs. Conversely, we found very low membrane-based receptor signaling, exhibiting <1% of the total receptor signaling for these eight RTKs. Moreover, we found that nuclear translocation, mechanistically, requires late endosomal transport; when we blocked receptor trafficking from late endosomes to the nucleus we found a 57% reduction in nuclear translocation. In summary, our research has elucidated the significance of endocytic vesicles, late endosomes and the nucleus in RTK signal propagation. 

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5. Cell isolation via spiral microfluidics and the secondary anchor targeted cell release system

   https://doi.org/10.1002/aic.16844  

   Ansari, Ali; Schultheis, Kinsey; Patel, Reema; Al‐Qadi, Kareem I.; Chen, Si; Jensen, Cassandra R.; Schad, Samantha R.; Weddell, Jared C.; Vanka, Surya P.; Imoukhuede, P. I. (November 2019, AIChE Journal)

   Abstract Precision medicine requires high throughput cell isolation and measurement that maintains physiology. Unfortunately, many techniques are slow or alter cell biomarkers cells. This necessitates new approaches, which we achieve by integrating affinity‐based cell isolation with spiral microfluidics. We characterize the device via computational simulations, predicting wall shear stress within an order of magnitude of arterial wall shear stress (~0.2 Pa). We identify that poly‐l‐lysine supplementation preserves cell geometry and improves cell release. We demonstrate preservation of angiogenic biomarker concentrations, measuring 1,000–2,000 vascular endothelial growth factor receptor‐1 per human umbilical vein endothelial cell, which is in line with the previously reported measurements. We attain 76.7 ± 9.0% release of captured cells by integrating thermophoresis and optimizing buffer residence time. Ultimately, we find that combining affinity‐based cell isolation (secondary anchor targeted cell release) with spiral microfluidics offers a fast, biomarker preserving approach needed to individualize medicine. 

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