Splicing Factor Kinase SRPK1 inhibition in Monocyte: A Novel Therapeutic Approach for Peripheral Arterial Disease

Peripheral arterial disease (PAD) is a complication of systemic atherosclerosis, affecting over 10 million people in the United States alone (1). Diabetes, a significant risk factor for PAD, significantly worsens outcomes, increasing the likelihood of developing critical limb threatening ischemia, which has a poor prognosis, high rates of limb amputation, and elevated mortality. Currently, no effective medical therapies are available to induce angiogenesis and promote perfusion recovery in patients with advanced PAD. Clinical trials aimed at boosting vascular endothelial growth factor (VEGF)-A levels, a potent proangiogenic growth factor, have failed to improve angiogenesis or perfusion recovery in these patients (1). Thus, there is an urgent need to identify novel molecular targets for therapeutic intervention. In this issue of the American Journal of Physiology, Bhalla et al. discover the serine-arginine protein kinase 1 (SRPK1) as a critical modulator of VEGF-A splicing in monocytes, revealing a novel mechanism contributing to impaired collateralization in obese PAD (Figure) (2). This compelling work positions SRPK1 inhibition as a promising therapeutic strategy for restoring angiogenic balance in diabetic PAD patients.

Figure: Role of SRPK1 in monocyte for driving VEGF-A splicing to anti-angiogenic isoform VEGF-A₁₆₅b and Macrophage polarization in PAD, particular with metabolic disease.

In PAD, especially in the context of metabolic diseases such as type 2 diabetes or obesity, increased expression of the anti-angiogenic VEGF-A₁₆₅b isoform (i.e., a decreased VEGF-A₁₆₅a/VEGF-A₁₆₅b ratio) in monocytes contributes to impaired collateralization. VEGF-A₁₆₅b upregulation has been observed in circulating monocytes of PAD patients, obese mouse models with peripheral ischemia, and coronary vascular disease models. A key mechanism underlying this VEGF-A splicing shift and impaired angiogenesis is the hyperactivation of the Wnt5a signaling pathway in monocytes. Notably, soluble-frizzled-related-protein-5 (Sfrp5) serves as an endogenous inhibitor of Wnt5a. In monocytes, Wnt5a activates SRPK1 via Ror/Fzd, which induces the splicing to anti-angiogenic VEGF-A₁₆₅b by phosphorylating SRSF2 (?). This, in turn, binds to the distal splice site (DSS) in VEGF-A pre mRNA. Bhalla et al. demonstrated that inhibiting SPRK1, either pharmacologically or genetically in Sfrp5-deficient mice, monocytes overexpressing Wnt5a, or monocytes from obese mice, not only prevented splicing toward the anti-angiogenic VEGF-A₁₆₅b isoform but also induced a macrophage shift toward a M2-like, pro-regenerative phenotype. This shift ultimately contributes to improved angiogenesis, collateral vessel formation, and blood flow recovery. Schematic Created in BioRender. Fukai, T. (2025) https://BioRender.com/q11i143

VEGF-A exists as two functionally distinct isoforms, which are generated through alternative splicing of the pre-mRNA(4). The selection of different 3’ splice sites in exon 8 can yield either proangiogenic isoforms, such as VEGF-A₁₆₅a, or antiangiogenic isoforms, like VEGF-A₁₆₅b. The two isoforms differ in the C-terminal, with VEGF-A₁₆₅b exhibiting a six-amino-acid shift from CDKPRR (proangiogenic) to PLTGKD (antiangiogenic). The replacement of positively charged arginine residues in VEGF-A₁₆₅a with neutral aspartic acid and lysine in VEGF-A₁₆₅b is predicted to decrease VEGFR2 activation and angiogenesis. VEGF-A₁₆₅b acts as a partial agonist for VEGFR1 and VEGFR2, inhibiting VEGF-A₁₆₅a-induced angiogenesis while also protecting endothelial and epithelial cells from cytotoxic damage (4). This isoform binds VEGFR1 and VEGFR2 but does not interact with neuropilin or heparan sulfate proteoglycans, leading to partial activation of VEGFR2 without full kinase activation due to lack of phosphorylation of Tyr1054 in VEGFR2 (4) and inhibiting VEGFR1 signalling (3).

In the context of PAD, particularly in patients with type 2 diabetes (T2D) or obesity, overexpression of the anti-angiogenic VEGF-A₁₆₅b isoform in monocytes contributes to impaired collateralization. Studies have shown that VEGF-A₁₆₅b is upregulated in the circulating monocytes of PAD patients and in obese mouse models with peripheral ischemia, as well as in coronary vascular disease models (3, 6). Additionally, the use of VEGF-A₁₆₅b-selective antibodies has been shown to improve collateralization in these models. A key mechanism driving this impaired angiogenesis has been proposed to be the hyperactivation of the Wnt5a pathway in monocytes (Figure). Monocytes from T2D patients and in animal models of T2D or obese exhibit increased Wnt5a expression, while levels of soluble-frizzled-related-protein-5 (Sfrp5), an endogenous inhibitor of Wnt5a, are reduced in plasma from patients with obesity, T2D, or both (6, 9). Sfrp5^−/− mice exhibited significantly reduced blood flow recovery and angiogenesis following hind limb ischemia, a well-established model of collateralization. This impairment is also observed in monocytes overexpressing Wnt5a. Importantly, neutralizing antibodies against VEGF-A₁₆₅b can reverse these angiogenesis defects. Thus, selective inhibition of VEGF-A₁₆₅b in monocytes may offer a novel therapeutic approach for PAD patients with diabetes and ischemia.

Pre-mRNA splicing of VEGF-A involves Serine/arginine (SR) proteins such as serine arginine-rich splicing factor 1 (SRSF1) and its modulator, Serine-arginine protein-kinase-1 (SRPK1). SRPK1 regulates the splicing of proangiogenic VEGF-A₁₆₅a by phosphorylating SRSF1 (8), which promotes its nuclear translocation and binding to the proximal splice site in VEGF-A pre mRNA. In epithelial and cancer cells, SRPK1 knockdown or inhibition has been shown to reduce the selection of the VEGF-A₁₆₅a splice site, leading to decreased production of proangiogenic VEGF-A (8). SRPK1 inhibition has also proven effective as an anti-angiogenic strategy in retinal diseases such as retinal and choroidal neovascularization (7). However, the regulation of VEGF-A splicing in monocytes, especially in those from diabetic patients with cardiovascular disease, remained unexplored.

In this issue, Bhalla et al. present several significant and novel findings (Figure) (2). First, a key discovery of this study is that SRPK1-mediated regulation of VEGF splicing is cell-type-specific. While previous research has primarily focused on VEGF splicing in epithelial and cancer cells, this study provides the first direct evidence that monocyte-specific VEGF splicing follows a distinct regulatory mechanism. Using co-culture models with monocytes from PAD patients and endothelial cells, the authors demonstrated that conditioned medium from PAD patient-derived monocytes inhibited endothelial cell migration. This inhibition was reversed not only by an anti-VEGF-A₁₆₅b antibody but also by the SRPK1 inhibitor SPHINX31, which shifts VEGF splicing from the anti-angiogenic VEGF-A₁₆₅b isoform to the pro-angiogenic VEGF-A₁₆₅a isoform. These findings challenge established paradigms and introduce new possibilities for cell-specific therapeutic interventions in ischemic vascular diseases.

Second, this study highlights the translational significance of SRPK1 inhibition, demonstrating its ability to enhance revascularization in multiple preclinical PAD models, particularly in T2D and obese patients (Figure) (2). In a high-fat/high-sucrose diet-induced obese mouse model of PAD, impaired blood flow recovery was restored in monocyte-specific SRPK1 knockout mice. Mechanistically, hyperactivation of the Wnt5a pathway in monocytes has been proposed as a key contributor to impaired collateralization in metabolic syndrome patients (6, 9). In T2D and obesity, circulating monocytes exhibit a proinflammatory phenotype and increased VEGF production, yet display reduced responsiveness to VEGF, a paradoxical phenomenon referred to as VEGF resistance (10). This paradox may, in part, be explained by the elevated expression and activity of Wnt5a observed in monocytes from diabetic patients and in mouse and rat models of T2D (9). Notably, Sfrp5, an endogenous Wnt5a inhibitor, is significantly reduced in plasma from individuals with obesity, T2D, or both. Sfrp5^−/− mice exhibit impaired ischemia-induced blood flow recovery and angiogenesis. Similarly, mice with monocytic lineage-specific Wnt5a overexpression demonstrate impaired angiogenesis despite a marked upregulation of VEGF-A expression, suggesting a dysfunctional proangiogenic response (6). Both Sfrp5-deficient and Wnt5a-overexpressing monocyte models exhibited increased VEGF-A₁₆₅b expression in monocytes, which was associated with reduced revascularization in the hindlimb ischemia model (6). The authors showed that inhibiting SRPK1 either pharmacologically or genetically in monocytes restored blood flow recovery in these models of impaired angiogenesis, including Sfrp5-deficient mice, mice with Wnt5a-overexpressing monocytes, and mice with obesity-induced metabolic dysfunction. These finding strongly suggest that targeting SRPK1 in monocytes could be a promising therapeutic approach to restore angiogenic balance in PAD patients.

Third, the authors found that pharmacological or genetic inhibition of SRPK1 in monocytes overexpressing Wnt5a or in monocytes from obese mice induced a shift in macrophage toward an M2-like, pro-regenerative phenotype (Figure) (2). These findings suggest that SRPK1 plays a critical role in macrophage polarization, fostering a more reparative, pro-arteriogenic, and less inflammatory macrophage phenotype. In addition, the authors observed upregulation of both SRPK1 and its target SRSF1 in ischemic tissue from Sfrp5^−/− mice, further supporting the link between Wnt5a signaling and SRPK1 activity. SRPK1 phosphorylates SR-rich sequences in SR proteins such as SRSF1. While prior studies have established SRSF1 as a primary SRPK1 target in epithelial cells, it is conceivable that in monocytes, SRPK1 may also phosphorylate SRSF2 or other SRSF proteins, given that SRSF2 has been implicated in the regulation of VEGF splicing in monocytes (6). Collectively, these findings underscore the importance of SRPK1 in monocyte-driven angiogenesis and identify it as a promising target for novel therapeutic interventions in PAD and related vascular disorders.

Despite these promising findings, several limitations warrant further investigation. First, VEGF splicing regulation is highly cell-type- and environment-dependent. The current findings in monocytes contrast with the well-documented effects of SRPK1 inhibition in other cell types, including epithelial and cancer cells, where SRPK1 inhibition shifts VEGF splicing toward the anti-angiogenic VEGF-A₁₆₅b isoform (7, 8). Notably, the diabetic environment alone does not appear to dictate whether SRPK1 promotes VEGF-A₁₆₅a or VEGF-A₁₆₅b splicing, as previous studies have shown that SRPK1 inhibition in diabetic retinal epithelial cells leads to VEGF-A₁₆₅b production (7), whereas in diabetic monocytes, it favors VEGF-A₁₆₅a. This highlights the need for a deeper understanding of how tissue- and developmental-stage-specific SRPK1 knockout influences tissue function. Generating conditional knockout models in tissues where VEGF isoform regulation differs, such as retinal epithelial cells (8), and retinal neurons (7), could provide critical insights into whether SRPK1-mediated isoform switching impacts conditions like diabetic nephropathy or retinopathy.

Additionally, further studies are needed to elucidate the precise mechanisms linking Wnt5a activation with SRPK1 activity, as well as the interplay between SRPK1 activity, macrophage phenotype, and VEGF-A splicing. Second, SRPK1’s role in angiogenesis may extend beyond the regulation of VEGF-A₁₆₅a and VEGF-A₁₆₅b ratios. Alternative splicing of VEGFR1 variants has also been shown to be mediated by SRPK1 via SRSF3 (5), and this mechanism should be investigated using SRPK1 knockout models. Third, while the study provides compelling evidence for SRPK1’s role in monocyte-driven angiogenesis, the long-term systemic effects of SRPK1 inhibition remain unclear. Future studies should assess potential off-target effects, particularly in other cell types where SRPK1 plays a role in VEGF regulation. Finally, although multiple mouse models were utilized, direct validation in larger animal models or early-phase clinical trials is lacking. Addressing these gaps will be essential for the successful translation of SRPK1-targeted therapies into clinical practice.

This study has significant therapeutic implications. The identification of SRPK1 as a modifiable regulator of monocyte-driven angiogenesis opens new avenues for targeted therapeutic strategies. Inhibiting SRPK1 offers a promising precision medicine approach for PAD by promoting a pro-angiogenic VEGF splicing profile while avoiding the off-target effects typically associated with exogenous VEGF delivery. Moreover, the ability of SRPK1 knockout to restore blood flow in obese mice highlights its potential application in treating vascular dysfunction related to metabolic syndrome. Future research should focus on evaluating the clinical feasibility of SRPK1 inhibitors in PAD patients, particularly their long-term safety and efficacy. Additionally, understanding how environmental factors such as hypoxia and inflammation influence SRPK1-mediated VEGF splicing will be essential for optimizing therapeutic interventions. Identifying biomarkers to stratify patient populations most likely to respond to SRPK1 inhibition could further enhance its clinical relevance and impact.

In summary, this study significantly advances our understanding of monocyte-specific VEGF splicing and its influence on angiogenesis in PAD. By identifying SRPK1 as a crucial regulator of collateral vessel formation, it not only deepens our mechanistic insights but also highlights SRPK1 as a potential therapeutic target. However, translating these findings into clinical applications requires overcoming challenges such as evaluating systemic effects and validating efficacy in human subjects. As research continues to refine the therapeutic potential of SRPK1 inhibitors, this discovery offers promising prospects for improving outcomes in patients with PAD and other vascular disorders.