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Background Angiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression. Results By combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients. Conclusions We demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-021-01103-3.

Additional File 2: Figure S2. Schematic representation of VEGFR1 and VEGFR1 splice variants. sVEGFR1-ex15a results from activation of a cryptic 3’-splice acceptor site upon the use of an alternative polyadenylation site in the latter half of intron 14. sVEGFR1-i13 short and sVEGFR1-i13 long result from alternative polyadenylation at different sites in intron 13 to yield mRNAs encoding the same 867 amino acid sVEGFR1 protein isoform, but with either a 17 or 4146 nt 3’-UTR region. sVEGFR1-ex12 retains an alternative last exon (exon 12). The location of forward and reverse primers used in RT-PCR analyses are indicated as black arrows on each transcript. siRNA sequences target exon 12 for sVEGFR1-ex12 and the junction between exon 13 and retained intron 13 for sVEGFR1-i13. (PPTX 68 kb)

Additional File 1: Figure S1. Effects of FGFR (AZD4547) and SRPK1 (SRPIN340, SPHINX31) inhibitors on VEGF165b protein and total VEGF-A, VEGF121, VEGF165 and VEGF189 mRNA levels in endothelial cells treated or not with FGF-2. (a) Representative VEGF165b immunoblots in HUVEC and HDMEC treated or not (NT) for 72 hours with 3nM FGF-2 in the presence or absence of 10nM AZD4547 (FGFRinh), 10μM SRPIN340 or 5μM SPHINX31 as indicated. GAPDH was used as a loading control. Representative immunoblots of two (HUVEC) and three (HDMEC) independent experiments are presented. (b) HDMEC cells were treated (FGF) or not (NT) with 3nM FGF-2 for 72 hours in the presence or absence of 5μM SPHINX31 (SPH31) or 10μM SRPIN340 as indicated. Graphs represent mean values ± SD of normalized expression of each transcript according to GAPDH mRNA level in 3 independent experiments. For each transcript, the fold change was calculated with value 1 assigned to the normalized expression value obtained in the non treated (control) condition. Unpaired t test, *p

Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.

Heterozygous missense or in-frame insertion/deletion mutations in complement 1 subunits C1r and C1s cause periodontal Ehlers-Danlos Syndrome (pEDS), a specific EDS subtype characterized by early severe periodontal destruction and connective tissue abnormalities like easy bruising, pretibial haemosiderotic plaques, and joint hypermobility. We report extensive functional studies of 16 C1R variants associated with pEDS by in-vitro overexpression studies in HEK293T cells followed by western blot, size exclusion chromatography and surface plasmon resonance analyses. Patient-derived skin fibroblasts were analyzed by western blot and Enzyme-linked Immunosorbent Assay (ELISA). Overexpression of C1R variants in HEK293T cells revealed that none of the pEDS variants was integrated into the C1 complex but cause extracellular presence of catalytic C1r/C1s activities. Variants showed domain-specific abnormalities of intracellular processing and secretion with preservation of serine protease function in the supernatant. In contrast to C1r wild type, and with the exception of a C1R missense variant disabling a C1q binding site, pEDS variants had different impact on the cell: retention of C1r fragments inside the cell, secretion of aggregates, or a new C1r cleavage site. Overexpression of C1R variants in HEK293T as well as western blot analyses of patient fibroblasts showed decreased levels of secreted C1r. Importantly, all available patient fibroblasts exhibited activated C1s and activation of externally added C4 in the supernatant while control cell lines secreted proenzyme C1s and showed no increase in C4 activation. The central elements in the pathogenesis of pEDS seem to be the intracellular activation of C1r and/or C1s, and extracellular presence of activated C1s that independently of microbial triggers can activate the classical complement cascade.