Fast in vitro protocol for the visualization and quantitative high-throughput analysis of sprouting angiogenesis by confocal microscopy.

We describe an optimized, cost-effective, reproducible, and robust protocol to study sprouting angiogenesis in glass-bottom 96-well plates by confocal microscopy, ideal for screening of drug or shRNA libraries. Effective and stable knockdown of gene expression in primary endothelial cells is achieved by lentiviral transduction. Dynamic behavior of individual cells and fluorescent proteins is analyzed by time-lapse imaging, while competitive advantages in tip cell formation are assessed using mixtures of differentially labeled cell populations. Finally, we present a macro for high-throughput analysis. For complete information on the use and execution of this protocol, please refer to van der Bijl et al. (2020) and Kempers et al. (2021).

The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis.

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

Variation in platelet-rich plasma compositions used for wound healing indications.

At present, the role of platelet-rich plasma (PRP) in burn and wound management is undefined. We present some of the evidence for PRP in wound healing and other medical fields. Currently, the high variation in product composition, mode and timing of application prevent a clear definition of the position of PRP in wound healing. Perspectives on solving these issues are described.

Reciprocal integrin/integrin antagonism through kindlin-2 and Rho GTPases regulates cell cohesion and collective migration.

Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding ß1 and ß3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal ß1/ß3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal ß1/ß3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.

MKL1 deficiency results in a severe neutrophil motility defect due to impaired actin polymerization.

Megakaryoblastic leukemia 1 (MKL1) promotes the regulation of essential cell processes, including actin cytoskeletal dynamics, by coactivating serum response factor. Recently, the first human with MKL1 deficiency, leading to a novel primary immunodeficiency, was identified. We report a second family with 2 siblings with a homozygous frameshift mutation in MKL1. The index case died as an infant from progressive and severe pneumonia caused by Pseudomonas aeruginosa and poor wound healing. The younger sibling was preemptively transplanted shortly after birth. The immunodeficiency was marked by a pronounced actin polymerization defect and a strongly reduced motility and chemotactic response by MKL1-deficient neutrophils. In addition to the lack of MKL1, subsequent proteomic and transcriptomic analyses of patient neutrophils revealed actin and several actin-related proteins to be downregulated, confirming a role for MKL1 as a transcriptional coregulator. Degranulation was enhanced upon suboptimal neutrophil activation, whereas production of reactive oxygen species was normal. Neutrophil adhesion was intact but without proper spreading. The latter could explain the observed failure in firm adherence and transendothelial migration under flow conditions. No apparent defect in phagocytosis or bacterial killing was found. Also, monocyte-derived macrophages showed intact phagocytosis, and lymphocyte counts and proliferative capacity were normal. Nonhematopoietic primary fibroblasts demonstrated defective differentiation into myofibroblasts but normal migration and F-actin content, most likely as a result of compensatory mechanisms of MKL2, which is not expressed in neutrophils. Our findings extend current insight into the severe immune dysfunction in MKL1 deficiency, with cytoskeletal dysfunction and defective extravasation of neutrophils as the most prominent features.

Allogeneic platelet-rich plasma (PRP) is superior to platelets or plasma alone in stimulating fibroblast proliferation and migration, angiogenesis, and chemotaxis as relevant processes for wound healing.

BACKGROUND: Platelet-rich plasma (PRP) is frequently used in the treatment of acute and chronic wounds. One of the major problems concerning the use of PRP is the absence of a well-characterized and standardized product, which leads to a high variety in study outcomes. Therefore, more studies on the composition and standardization of PRP in wound healing are needed. STUDY DESIGN AND METHODS: Platelet concentrates derived from healthy blood donors were made in plasma (PC-plasma) or platelet additive solution (PC-PAS). The effects of PC-plasma, PC-PAS, and plasma were then tested on proliferation, differentiation, and migration of fibroblasts, as well as sprouting of endothelial cells in fibrin gels and chemotaxis of white blood cells (WBCs). RESULTS: PC-plasma stimulates the migration and proliferation of human dermal fibroblasts more than plasma or platelets alone. Furthermore, platelet factors decrease the expression of α-smooth muscle actin in dermal fibroblast cultures. PC-plasma also stimulates sprouting of endothelial cells. Finally, PC-plasma also acts as a strong chemoattractant for WBCs. CONCLUSIONS: Allogeneic PC-plasma has beneficial effects on various aspects of wound healing in vitro and is superior to plasma or platelets alone. PC-plasma is an attractive candidate for further in vivo evaluation.

Activation, function and content of platelets in burn patients.

Burn injury has severe impact on the physiologic homeostasis. Platelet counts show a distinct course post-burn injury, with a nadir at day 3 followed by a thrombocytotic period with at peak at day 15, with a gradual return to normal. So far, it is unknown how the functionality and activational status of platelets develop post burn. In this study, we investigated if the function, activation and growth factor content of platelets of burn patients are affected and how this evolves in time. Six burn patients with over 15% total burned surface area were followed during 1 month. Standard hematological and coagulation analyses, thromboelastography (TEG), platelet-function analyzer-100 (PFA), several platelet activation parameters (CD62P-CD63, AnnexinV) and growth factors (TGFb1, VEGF, PDGF-AB/BB, EGF, TGFb2, FGF-2, PDGF-AA) analyses were performed. TEG analyses showed procoagulant changes. PFA-100 analyses were nearly all within normal range. CD62P and CD63 and Annexin-V indicated no clear activation of platelets. Growth factor content followed the same course as the platelet count, reflecting a constant growth factor per platelet ratio. Concluding, platelets post burn-injury appears to be functional and not overly activated. However, burn patients seem to remain in a procoagulant state for an extensive period, which may impact their pathology.