The role of VEGF₁₆₅b in pathophysiology

Abstract

Anti-angiogenic vascular endothelial growth factor A (VEGF)₁₆₅b and pro-angiogenic VEGF₁₆₅ are generated from the same transcript, and their relative amounts are dependent on alternative splicing. The role of VEGF₁₆₅b has not been investigated in as much detail as VEGF₁₆₅, although it appears to be highly expressed in non-angiogenic tissues and, in contrast with VEGF₁₆₅, is downregulated in tumors and other pathologies associated with abnormal neovascularization such as diabetic retinopathy or Denys Drash syndrome. VEGF₁₆₅b inhibits VEGFR2 signaling by inducing differential phosphorylation, and it can be used to block angiogenesis in in vivo models of tumorigenesis and angiogenesis-related eye disease. Recent reports have identified three serine/arginine-rich proteins, SRSF1, SRSF2 and SRSF6, and studied their role in regulating terminal splice-site selection. Since the balance of VEGF isoforms is lost in cancer and angiogenesis-related conditions, control of VEGF splicing could also be used as a basis for therapy in these diseases.

Cells of pluricellular organisms require access to the circulation to subsist. Blood vessels carry the nutrients and oxygen necessary for the survival of these cells, and remove metabolic waste and carbon dioxide away from them. In the adult, the formation of new blood vessels is a tightly regulated phenomenon, becoming activated during brief periods of time such as wound healing or the female reproductive cycle, and subsequently inhibited.¹ In a physiological context, stimulators and inhibitors apply positive and negative influences on blood vessel formation to ensure that it is limited to the demands of the growing tissue. This balance is lost in many pathological conditions, leading to an angiogenic phenotype. Vascular endothelial growth factor (VEGF) is a key contributor to this process in health and disease. The VEGF family of growth factors includes VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C and VEGF-D, being VEGF-A the most important member of the family.

The human vegf-a gene contains eight exons and seven introns.² Alternative splicing and proteolytic processing of VEGF-A produces various isoforms with distinct biological activities (Fig. 1A).^3-8 The particular splicing event in the terminal exon creates two whole families of isoforms: the classic pro-angiogenic VEGF_xxx isoforms, generated by proximal splice-site (PSS) selection, and the more recently described anti-angiogenic VEGF_xxxb isoforms, formed by distal splice-site (DSS) choice, 66 bases downstream (Fig. 1B).⁹ This process gives rise to two families of polypeptides with the same size but different sequence at the C-terminus, maintaining the two of them the dimerization and receptor binding domains unchanged.³ Exon 8a encodes Cys-Asp-Lys-Pro-Arg-Arg instead of the Ser-Leu-Thr-Arg-Lys-Asp sequence encoded by exon 8b. Therefore, the basic properties conferred by the two arginines present in 8a are altered, resulting in the neutral charge conferred by the residues lysine and aspartic acid, and loss of one disulfide bond. These conformational changes result in the inability of VEGF_xxxb to bind the VEGFR2 co-receptor neuropilin 1 (NRP1), and have been proposed as the cause for the differences in downstream signaling (Fig. 1C).^10,11

Figure 1.vegf-a. (A) Gene structure. TSS is the transcriptional start site. (B) mRNA species. Alternative splicing of the vegf-a gene in the terminal exon results in two families of isoforms: the pro-angiogenic VEGF_xxx and the anti-angiogenic VEGF_xxxb isoforms. AUG, the start site for translation; UTR, the untranslated region; pA, the polyadenylation site. (C) Protein structure of the two major isoforms of each family, VEGF₁₆₅ and VEGF₁₆₅b. C-terminal splicing leads to an alternative last six amino acids (CDKPRR or SLTRKD). The isoforms are termed according to the amino acid number of the resulting protein (_xxx). HSPG, heparin sulfate proteoglycan; R1, VEGF Receptor 1; R2, VEGF Receptor 2 (from ref. 29).

VEGF₁₆₅b Physiological Expression and Function

VEGF₁₆₅b, the major anti-angiogenic isoform, was the first member of the VEGF_xxxb family to be described³ and is the most studied member so far. VEGF₁₆₅b mRNA was first isolated and cloned from human renal cortex tissue, and subsequently identified in other human tissues. In fact, anti-angiogenic VEGF isoforms have been reported to represent a predominant proportion of the total VEGF protein found in normal human tissues such as vitreous fluid, circulating plasma, urine, renal cortex and glomeruli, colonic epithelium, bladder, smooth muscle, lung and pancreatic islets.^9,12,13 On the contrary, in placenta, a tissue where angiogenesis takes place under physiological conditions, VEGF_xxxb represents only 1.5% of total VEGF.¹² In vitro, VEGF_xxxb also predominates in primary cultured cells, such as differentiated podocytes or retinal pigmented epithelial cells. VEGF_xxxb isoforms have also been reported in other species including pig,¹⁴ rabbit, mouse¹⁵ and rat,^16,17 although its relative expression has been found to be decreased from humans to lower mammals, suggesting that the inclusion level of exon 8b, as well as the complexity of the regulatory mechanism of its splicing, increases through the evolutionary process from mouse to human.¹⁵ The splice site is not conserved in lower vertebrates,³ and there is no information on the existence of VEGF₁₆₅b outside mammals.

VEGF₁₆₅ binding to VEGFR2 and NRP1 results in dimerization of the receptor and rotation of its intracellular domain, leading to its autophosphorylation (Fig. 2A).¹⁸ By contrast, this full rotational change is predicted to not occur when VEGF₁₆₅b binds VEGFR2, resulting in inefficient autophosphorylation of the receptor (Fig. 2B). In fact, the preference of VEGF₁₆₅b toward the various tyrosine phosphorylation sites of the receptor is different than that observed after VEGF₁₆₅ activation. Particularly, tyrosine 1054¹⁸ in the kinase regulatory site is not phosphorylated following VEGF₁₆₅b-VEGFR2 binding. Once VEGF₁₆₅b has bound the receptor, what happens downstream? Little is known about the mechanisms of VEGF₁₆₅b signaling. VEGF₁₆₅b stimulates VEGFR2, ERK1/2 and Akt phosphorylation in endothelial cells.^8,19 However, VEGF₁₆₅b-induced phosphorylation of ERK1/2 has been observed to be more transient and weaker than that promoted by VEGF₁₆₅.^4,8,18,19

Figure 2. VEGF₁₆₅b and VEGF₁₆₅ interaction with VEGF receptor 2 (VEGFR2). (A) The VEGFR2 binding site of VEGF₁₆₅ interacts with the VEGFR2 extracellular domain. VEGF₁₆₅ functions as a dimer and promotes the formation of VEGFR2 dimers resulting in activation of the kinase domains via the phosphorylation of its tyrosine residues. Robust tyrosine phosphorylation results in the activation of angiogenic signaling pathways. (B) VEGF₁₆₅b binds the VEGFR2 binding site with equal affinity to VEGF₁₆₅ but does not bind neuropilin 1 (NRP1) co-receptor. The C-terminus of VEGF₁₆₅b is neutral and there is insufficient torsional rotation for complete tyrosine phosphorylation, although weak phosphorylation can occur (adapted from ref. 11)

VEGF₁₆₅ and VEGF₁₆₅b appear to have contrasting physiological roles. The functions of VEGF₁₆₅b are summarized in Table 1.

Table 1. VEGF₁₆₅b physiological functions.

Function	Effect	References
Proliferation and migration	VEGF165b does not stimulate endothelial cell proliferation, and inhibited proliferation in the presence of VEGF165 and inhibits VEGF165-stimulated migration of endothelial cells	3 and 35
	Endogenous overexpression of VEGF165b inhibits migration and proliferation of endothelial cells	8
Angiogenesis	VEGF165b inhibited endothelial cell tube formation in vitro	30
	VEGF165b inhibited physiological angiogenesis in mammary tissue in transgenic mice and rabbit cornea	8 and 45
	VEGF165b can specifically inhibit VEGF165-induced angiogenesis in mouse skin, rat mesentery, rabbit cornea, and chick chorioallantoic membrane	4, 8, 28, 30 and 31
	VEGF165b prevents angiogenesis and helps maintaining the established vascular system in the fetal human eye	54
Survival and cytoprotection	VEGF165b acts as a survival factor in different cell types including endothelial cells, podocytes, retinal pigmented cells or colonic adenoma cells, but not in cone photoreceptors	12, 22, 28, 35 and 55
	VEGF165b could protect the retina from ischemic damage	35
Permeability and vasodilatation	VEGF165b does not increase chronic microvascular permeability	56
	Inhibition of VEGFxxxb reduces glomerular endothelial and VEGF165-induced permeability in vitro	12
	VEGF165b did not stimulate vasodilatation ex vivo, and inhibited that mediated by VEGF165	57
Gonadal development	Inhibition of VEGFxxxb in developing testis stimulates vascular development similarly to VEGF164 addition	58
	Inhibition of VEGFxxxb in developing ovary results in follicle progression similarly to VEGF164 stimulation, and abnormal ovarigenesis due to enhanced neovascularization	16

Regulation of VEGF₁₆₅b Expression

VEGF₁₆₅b expression can be regulated by growth factors, including insulin-like growth factor 1 (IGF-1), tumor necrosis factor α (TNFα) and transforming growth factor β (TGFβ) in retinal pigmented epithelial cells, podocytes and dermal microvascular endothelial cells (MVECs). IGF-1 and TNFα have been shown to modulate VEGF splicing preferentially at the PSS increasing the expression of pro-angiogenic VEGF isoforms. In podocytes, IGF-mediated VEGF_xxx upregulation occurs via stimulation of protein kinase C (PKC), which in turn activates the serine/arginine protein kinase 1 (SRPK1). Finally, SRPK1 phosphorylates the serine/arginine-rich splicing factor 1 (SRSF1), also known as ASF/SF2. Once activated, SRSF1, previously identified as an oncogene due to its transforming capacity in vitro and in vivo,²⁰ is able to bind VEGF pre-mRNA in the area adjacent to the PSS promoting VEGF_xxx expression (Fig. 3A).^21,22

Figure 3. Regulation of VEGF alternative splicing in the terminal exon.

On the other hand, TGFβ upregulates anti-angiogenic VEGF isoforms most likely through stimulation of the p38 MAPK pathway and successive downstream activation of Clk/sty kinases, which results in phosphorylation of the serine/arginine-rich splicing factor 6 (SRSF6), also known as SRp55. SRSF6 binds directly to VEGF pre-mRNA in the exon 8b region favoring DSS selection and therefore, VEGF_xxxb expression (Fig. 3B).^22,23 Finally, transcription factor 1 (E2F1) also favors the expression of the anti-angiogenic isoforms in human lung carcinoma cell lines through upregulation of another serine/arginine-rich splicing factor, SRSFR2, also known as SC35 (Fig. 3B).²⁴ SRSF2 overexpression is able to mimic the E2F1-induced VEGF₁₆₅b expression in vitro, and decreases tumor neovascularization in vivo. In contrast, a direct correlation between VEGF_xxx and E2F1 has been observed in colorectal cancer specimens²⁵ suggesting that the regulation of vegf-a splicing by E2F1, and in general by SR splicing factors in human tumor cells, could be more complex and might depend on different factors such as the cell type, the amount of the different SR proteins, or other upstream signals.

VEGF₁₆₅b in Pathology

The role of VEGF₁₆₅b in cancer

Vasculature not only provides tumors with an adequate blood supply, but it offers a route for tumor cells to metastasize.²⁶ An upregulation of the pro-angiogenic VEGF_xxx variants has been widely reported in human tumors. This upregulation brings about a loss in the balance of isoforms, which causes a drop in the proportion of VEGF_xxxb levels. Indeed, VEGF_xxxb is downregulated in several adult epithelial cancer types including melanoma,²⁷ renal⁸ or colorectal carcinoma.^25,28 An imbalance of the expression of the two VEGF families of isoforms has also been observed in the pediatric cancer neuroblastoma.²⁹ In contrast, VEGF_xxxb is upregulated in intraductal breast carcinoma.¹⁹

Overexpression of VEGF₁₆₅b has been reported to delay the growth of melanoma,⁸ kidney,³⁰ colon,²⁸ prostate and Ewing sarcoma tumors in mice.³¹ Recombinant human VEGF₁₆₅b (rhVEGF₁₆₅b) treatment in vivo also has a growth-inhibitory effect in nude mice xenograft models of melanoma,⁸ prostate cancer, renal cell carcinoma, Ewing’s sarcoma,³¹ colorectal carcinoma²⁸ and neuroblastoma.²⁹ Likewise, rhVEGF₁₆₅b parenteral administration reduces the growth of melanoma metastases.¹³ These studies highlight the potential role of VEGF₁₆₅b as an anti-cancer agent. However, a proper selection of patients that can benefit from this treatment is essential, as rhVEGF₁₆₅b-based therapies are likely to be effective only in tumors with high VEGF expression vs. those with low VEGF levels, which must rely on other pro-angiogenic factors for their growth.¹⁹

Finally, it has been shown that the VEGF_xxx/VEGF_xxxb ratio has an effect on the sensitivity of tumors to bevacizumab, an anti-VEGF antibody licensed for use in cancer treatment: as both VEGF₁₆₅ and VEGF₁₆₅b can bind bevacizumab with a similar affinity, the presence of VEGF_xxxb can inhibit the effect of this drug through reducing the amounts of antibody available to bind VEGF_xxx.²⁸ Hence, despite having a slower growing rate, tumors with high concentrations of VEGF_xxxb might be more resistant to this particular therapy. One alternative treatment option for these tumors would be to use VEGF_xxx-specific antibodies, which would inhibit exclusively the pro-angiogenic isoforms. Another possibility would be to develop molecules able to favor the DSS selection, and thus increase the endogenous expression of VEGF_xxxb. Indeed, it has been shown that knocking down SRPK1 in a colorectal tumor cell line sensitive to the anti-angiogenic actions of VEGF₁₆₅b, decreases tumor growth in vivo.³²

The role of VEGF₁₆₅b in eye disease

A high expression of VEGF₁₆₅b has been reported in human eye tissues, such as the retina or vitreous fluid,⁹ and in the rodent eye.^17,33 However, VEGF₁₆₅b is relatively downregulated in vitreous fluid of diabetic retinopathy patients, resulting in a switch to an angiogenic phenotype.⁹ In fact, proliferative diabetic retinopathy is characterized by an abnormal growth of new blood vessels into the vitreous fluid of the eye, stimulated by hypoxia-induced VEGF expression. In a mouse model of oxygen-induced retinopathy (OIR), the development of neovascularization in the retina correlated with an increase of total VEGF, and a specific decrease of VEGF₁₆₅b, indicating a pro-angiogenic shift.³³ In this OIR mouse model, intraocular injection of rhVEGF₁₆₅b also diminished pre-retinal neovascularization yet permitting the physiological angiogenesis in the inner retina to proceed normally.³⁴ This lack of intra-retinal neovascularization could be a consequence of the cytoprotective effects of VEGF₁₆₅b on endothelial cells.³⁵ Thus, treatments with VEGF₁₆₅b would not only inhibit neovascularization but still protect existing endothelial cells in a disease in which angiogenesis is frequently a consequence of endothelial cell loss and ischemia.³⁵ The VEGF balance is also altered in other angiogenic-associated eye conditions, including retinal vein occlusion³⁶ or neovascular age-related macular degeneration (AMD), and rhVEGF₁₆₅b acted as a powerful anti-angiogenic agent when injected either subcutaneously or intraocularly in a mouse model of AMD, where choroidal neovascularization was laser-induced.³⁷ These results, together with the cytoprotection that VEGF₁₆₅b confers to cultured retinal epithelial cells,³⁵ point to this anti-angiogenic isoform as a potential therapeutic agent in angiogenesis-associated eye diseases.

In contrast, in avascular eye diseases, including glaucoma,¹⁷ rhegmatogenous retinal detachment and proliferative vitreoretinopathy,³⁸ VEGF_xxxb has been found to be upregulated suggesting that, besides impeding neovascularization, VEGF₁₆₅b might play a role in the pathogenesis of these conditions that are not angiogenesis-related.

The role of VEGF₁₆₅b in renal disease

VEGF₁₆₅b expression has been reported in the glomeruli of human kidneys.¹³ However, whereas overexpression of VEGF₁₆₄ in mouse glomeruli leads to proteinuria, glomerular dysfunction and renal failure,³⁹ a transgenic mouse model where VEGF₁₆₅b was overexpressed in podocytes, did not have consequences on renal function. Nevertheless, closer observations of these podocytes showed decreased glomerular water permeability, which was dose-dependent.⁴⁰ In addition, ex vivo incubation of wild-type mouse glomeruli with VEGF₁₆₅b, decreased permeability to water,⁴¹ and constitutive overexpression of VEGF₁₆₅b could prevent the observed VEGF₁₆₄-driven increased water permeability and the changes in glomerular ultrastructure in vivo,⁴² indicating that maintaining an appropriate VEGF isoform balance is also important in the glomerular function of the kidney.

Changes in the balance of VEGF-A variants were also found to be important in Denys Drash syndrome, a urogenital disorder associated with nephropathy and high risk for Wilms tumors. While pro-angiogenic VEGF₁₆₅ was upregulated, VEGF₁₆₅b was completely abrogated in glomeruli of patients with Denys Drash syndrome (DDS) suggesting a switch in splicing.⁴³ The authors pointed out that aberrant expression of the WT1 gene may be involved in regulating the vegf-a gene expression, and is the cause of glomerulopathy in DDS. In fact, WT1 in wild-type podocytes could bind the SRPK1 promoter inhibiting its expression (Fig. 3B), whereas the mutated form of WT1 in podocytes isolated from a DDS patient did not bind this site, and SRPK1 expression was upregulated. Wild-type WT1 restoration decreased SRPK1 expression in these podocytes.^22,32 Nevertheless, WT1 is known for being able to both repress and activate its target genes, hence its transcription function is highly context-specific and can be modulated by several cofactors.⁴⁴ Thus, modifying vegf-a splicing by preventing SRPK1 activity may be therapeutically effective in DDS.

The role of VEGF₁₆₅b in fertility control and pregnancy-related conditions

A transgenic mouse model, whereby overexpression of VEGF₁₆₅b is driven by the mouse mammary tumor virus promoter, shows expression of VEGF₁₆₅b in tissues such as the ovary or the mammary tissue during mammary development of female mice. These mice had fewer blood vessels in the mammary tissue, an impaired alveolar vascular coverage of the fat pad and a reduced production of milk for nourishment of their pups, which died of starvation shortly after birth.⁴⁵ Whereas the litter size of wild-type females mated with transgenic males was normal, when transgenic females were mated with wild-type mice, significantly fewer pups were born. However, no developmental defects in the embryos were detected in culture which, together with the normal birth weight of the pups, indicated that the reduction in litter size could depend on ovarian function, as follicular development in the ovary is angiogenesis-dependent. Indeed, VEGF₁₆₅b overexpression in the mouse ovary caused an increase in the ovulatory cycle length and a delayed follicular development, resulting in fertility defects in these mice.⁴⁵ Additionally, VEGF₁₆₅b neutralization with an antibody specific for VEGF_xxxb, as well as treatment with the pro-angiogenic isoform VEGF₁₆₄, showed an enhanced vascular and follicular development in perinatal rat ovaries.¹⁶

Total VEGF levels are upregulated and VEGF_xxxb isoforms are expressed at even lower levels than usual in the placenta of patients with pre-eclampsia,⁴⁶ a condition in which the vasculature of pregnant women is vasoconstricted and shows higher permeability.⁴⁷ Circulating VEGF₁₆₅b levels are downregulated during the first trimester in pregnancies that will develop pre-eclampsia, making it a useful clinical marker for high risk of pre-eclampsia.⁴⁸ Although after the first trimester circulating VEGF_xxxb levels are normal in pre-eclamptic patients, pre-eclamptic plasma has been shown to increase vascular permeability. This increase could be inhibited by incubating the pre-eclamptic plasma with an antibody specific for VEGF_xxxb, suggesting that VEGF_xxxb is responsible for the enhanced vascular permeability in pre-eclamptic plasma.⁴⁷ Therefore, VEGF₁₆₅b plays a key role not only in reproductive biology, but also in the control of fertility and pre-eclampsia.

The role of VEGF₁₆₅b in systemic sclerosis

VEGF₁₆₅b upregulation has been reported in the skin and circulation of patients with systemic sclerosis (SSc), a chronic disease characterized by impaired angiogenesis and vascular repair that affects the skin and internal organs.^49,50 Cultured dermal microvascular endothelial cells (MVECs) isolated from SSc patients expressed higher levels of VEGF₁₆₅b and VEGFR2 than those from healthy individuals. In spite of this, VEGFR2 phosphorylation was reduced and angiogenesis, defective as a consequence. Furthermore, treatment with rhVEGF₁₆₅b, as well as treatment with conditioned media from SSc MVECs, inhibited VEGF₁₆₅-induced VEGFR2 phosphorylation and angiogenesis in healthy MVECs, and these anti-angiogenic effects could be neutralized by treatment with a VEGF₁₆₅b-blocking antibody.²³ Thus, neutralization of the elevated levels of VEGF₁₆₅b might represent a potential therapeutical approach to stimulate a proper angiogenic response in SSc patients.

In addition, the high VEGF₁₆₅b levels in SSc skin correlated with increased expression of TGFβ and SRSF6, and expression of VEGF₁₆₅b and SRSF6 was found to be increased and regulated by TGFβ in cultered SSc MVECs, indicating that in SSc, and other non-angiogenic disorders, favoring PSS rather than DSS selection might represent a potential therapeutic strategy to stimulate adequate angiogenesis.

Perspective

Every year there are more and more studies on VEGF₁₆₅b, and new conditions where VEGF₁₆₅b is identified as altered or contributory—for instance a polymorphisim in intron 7 that appears to regulate VEGF₁₆₅b expression predicts susceptibility to asthma.⁵¹ Since its discovery, over 50 publications have described VEGF₁₆₅b as an anti-angiogenic agent that is downregulated in cancer and other angiogenesis-related conditions such as diabetic retinopathy, and plays an important role in permeability regulation and renal disease, pre-eclampsia and fertility control. Nevertheless, there are researchers who are still skeptical about VEGF₁₆₅b predominance in human or even existence in mouse tissue.⁵² Indeed, despite being discovered in 2002, few reports on the splicing mechanism that gives rise to VEGF_xxxb and its effects on angiogenesis-related diseases have been published during the last decade, and its downstream signaling effects remain practically unreported. One of the reasons that could explain this lack of research is the difficulty of quantifying the absolute amounts of VEGF_xxx and VEGF_xxxb mRNA and protein.^52,53 The 3′UTR secondary structure of VEGF₁₆₅ and VEGF₁₆₅b transcripts is likely to be different, and result in differential reverse transcription efficiency. In fact, VEGF₁₆₅ has been shown to be preferentially amplified by RT-PCR when mixed at equal amounts or even in excess of VEGF₁₆₅b cDNA.⁵³ When VEGF was first identified, the importance of ruling out misamplification of VEGF₁₆₅ by VEGF₁₆₅b primers was highlighted.³ A recent publication reiterated the importance of primer design in avoiding annealing of the reverse primer to VEGF_xxx and amplification of a misprimed product,⁵² although in that report, the authors did not use positive control DNA to ensure that the primers act specifically, and amplify the VEGF_xxxb products efficiently. Mispriming is easily prevented by using primers that can simultaneously amplify both VEGF_xxx and VEGF_xxxb transcripts, and by subsequent sequencing of the obtained amplicons to confirm their nature, while using control DNA to account for differential amplification. Moreover, there is no commercially available VEGF_xxx-specific antibody, and the sensitivity of the currently existing VEGF_xxxb ELISA for the different isoforms and heterodimers of these isoforms has not been tested. The total VEGF ELISA, for example, underestimates the total VEGF levels as it has less affinity for VEGF₁₆₅b than for VEGF₁₆₅.²⁸ Therefore, further efforts need to be made to develop appropriate and reliable methods that allow the accurate quantification of both VEGF_xxx and VEGF_xxxb isoforms.

Growing evidence shows that vegf-a alternative splicing can be differentially regulated in conditions characterized by an excess of angiogenesis, such as cancer, or a deficit, such as SSc, but the control of this splicing event is still not well understood, and determining how different key splicing factors regulate an angiogenic splicing switch should be studied much more in depth over the next few years. The cellular machinery underlying the control of splice-site selection, and consequently which isoforms are being expressed to the detriment of others, represents a promising field for continuing investigations, taking into consideration that regulation of this splicing is not likely to be restricted to VEGF-A.

Glossary

Abbreviations:

AMD

age-related macular degeneration

DDS

Denys-Drash syndrome

DSS

distal splice site

IGF-1

insulin-like growth factor 1

MVECs

microvascular endothelial cells

NRP1

neuropilin 1

OIR

oxygen-induced retinopathy

PSS

proximal splice site

SRPK1

serine/arginine protein kinase 1

SRSF1

serine/arginine-rich splicing factor 1

SRSF2

serine/arginine-rich splicing factor 2

SRSF6

serine/arginine-rich splicing factor 6

SSc

systemic sclerosis

TGFβ

transforming growth factor beta

TNFα

tumor necrosis factor alpha

UTR

untranslated region

VEGF

vascular endothelial growth factor

VEGFR2

vascular endothelial growth factor receptor 2

WT1

Wilms tumor 1

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.