B55α Orchestrates AMPK/SIRT1/HIF-1α Signaling: VCE-005.1 as a Tissue-Selective Therapeutic Strategy for Ischemic Vascular Diseases

B55α Orchestrates AMPK/SIRT1/HIF-1α Signaling: VCE-005.1 as a Tissue-Selective Therapeutic Strategy for Ischemic Vascular Diseases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article B55α Orchestrates AMPK/SIRT1/HIF-1α Signaling: VCE-005.1 as a Tissue-Selective Therapeutic Strategy for Ischemic Vascular Diseases Isabel Lastres-Cubillo, María E. Prados, Juan J. Ferres-Serrano, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6319136/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Peripheral artery disease (PAD) and diabetic foot ulcers (DFUs) are chronic ischemic conditions characterized by endothelial dysfunction, impaired angiogenesis, and tissue hypoxia. The regulatory phosphatase subunit B55α (PP2A) modulates PHD2/HIF-1α axis, supporting vascular homeostasis and repair. Here, we investigated the mechanism and therapeutic potential of VCE-005.1, a selective B55α activator and PHD2 inhibitor, in PAD and DFU preclinical models. In human endothelial cells, VCE-005.1 activated B55α/AMPK/SIRT1/HIF-1α axis by inducing AMPK phosphorylation, elevating intracellular NAD⁺ levels, upregulating SIRT1 expression and enzymatic activity, stabilizing HIF-1α, and enhancing eNOS phosphorylation. VCE-005.1 also prevented oxidative stress–induced endothelial senescence by reducing p21 and restoring SIRT1 levels. In macrophages, it inhibited foam cell formation and induced apoptosis via PARP-1 fragmentation. In a murine critical limb ischemia (CLI) model, VCE-005.1 enhanced arteriogenesis, endothelial proliferation, and mature vessel formation in hypoxic muscle, while selectively upregulating angiogenic genes ( Vegf-A, Hgf, Epo ) and caveolin-1. Plasma biomarker analysis revealed that VCE-005.1 normalized markers of inflammation, endothelial dysfunction, apoptosis, vascular aging, and promoted neurovascular repair. In diabetic db/db mice, topical VCE-005.1 improved wound closure, re-epithelialization, collagen deposition, and microvascular density, while reducing neutrophil and macrophage infiltration. These effects correlated with localized induction of B55α and SIRT1 expression in endothelial and dermal papilla cells. These findings position VCE-005.1 as a promising tissue-selective therapeutic candidate for ischemic vascular diseases. By enhancing angiogenesis, preventing endothelial senescence, reducing cellular damage, and selectively targeting hypoxic tissues, VCE-005.1 may overcome the limitations of current pro-angiogenic therapies, offering new hope for patients with PAD and DFUs. B55α activators AMPK Sirtuin 1 angiogenesis diabetic foot Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Peripheral artery disease (PAD) is a chronic vascular disorder primarily caused by atherosclerosis, leading to arterial narrowing or occlusion, particularly in the lower extremities. This restriction in blood flow results in ischemia, manifesting as intermittent claudication, pain, and fatigue during physical activity [1]. If left untreated, PAD can progress to critical limb ischemia (CLI), a severe condition associated with non-healing ulcers, gangrene, and a significantly increased risk of amputation [2]. Globally, PAD affects over 200 million individuals, with its prevalence rising due to common risk factors such as smoking, diabetes, hypertension, and dyslipidemia [3]. Diabetes further exacerbates PAD, predisposing individuals to complications such as diabetic foot ulcers (DFUs). These ulcers develop in approximately 15–25% of diabetic patients during their lifetime and are frequently associated with vascular insufficiency and neuropathy. Notably, nearly 50% of DFU cases co-occur with PAD, leading to impaired wound healing, recurrent infections, and increased amputation rates [4]. The interplay between reduced blood flow, endothelial dysfunction, and impaired tissue repair in PAD and DFU results in chronic peripheral tissue hypoxia, posing a major clinical challenge [4, 5]. These conditions collectively contribute to significant morbidity, mortality, and healthcare costs, highlighting the urgent need for innovative therapeutic strategies targeting endothelial dysfunction and impaired vasculogenesis. In PAD and DFU, ischemia and hypoxia trigger compensatory pro-angiogenic mechanisms, notably via the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and growth factors such as vascular endothelial growth factor (VEGF). However, chronic endothelial dysfunction-driven by oxidative stress, inflammation, and impaired endothelial cell signaling-compromises the formation of functional blood vessels. Despite elevated VEGF expression, the damaged vascular microenvironment results in defective or insufficient angiogenesis, failing to restore adequate perfusion or support tissue regeneration [2, 6]. This underscores endothelial dysfunction as a critical barrier to effective angiogenic therapies in PAD and DFU. HIF-1α is a key transcriptional regulator of the cellular response to hypoxia, orchestrating the expression of pro-angiogenic factors such as VEGF, hepatocyte growth factors (HGF), and erythropoietin (EPO). Under hypoxic conditions, prolyl hydroxylases (PHDs) are inhibited, allowing HIF-1α stabilization and activation of genes essential for angiogenesis and vascular remodeling [7]. Hypoxic preconditioning, achieved through controlled exposure to low oxygen levels or hypoxia-mimetic compounds, has demonstrated potential benefits in vascular diseases by enhancing HIF-dependent pathways, improving tissue oxygenation, and promoting vascular repair [8, 9]. While this strategy holds promise for ischemic conditions like PAD and CLI [10, 11], its therapeutic potential in DFU remains largely unexplored. Recent research suggests that the regulatory subunit B55α (PPP2R2A) of protein phosphatase 2A (PP2A) could be a novel pharmacological target in vascular diseases. The B55α subunit dephosphorylates PHD2 at Ser125, reducing its activity and subsequently enhancing HIF-1α accumulation and signaling [12]. Additionally, B55α stabilizes endothelial cells (ECs), protecting them against oxidative stress and apoptosis during vascular remodeling [13]. This mechanism not only preserves endothelial integrity but also fosters angiogenesis, supporting new blood vessel formation. On the other hand, SIRT1, a NAD + -dependent deacetylase, is a pivotal regulator of vascular function, maintaining endothelial homeostasis and promoting vasodilation, angiogenesis, and tissue regeneration[14]. SIRT1 activity is tightly linked to AMPK, a master regulator of cellular metabolism and vascular function [15]. Dysregulation of the AMPK-SIRT1 axis contributes to endothelial dysfunction and impaired vascular repair in PAD and DFU [16], further supporting the therapeutic potential of AMPK-SIRT1-targeting compounds. Given its role in vascular homeostasis, PP2A/B55α activators such as VCE-005.1 and VCE-004.8 (Etrinabdione) emerge as promising candidates for therapeutic intervention in PAD, CLI, and DFU. While Etrinabdione is already in phase IIa clinical trial for PAD patients (clinicaltrials.gov: NCT06774040), VCE-005.1, a betulinic hydroxamate, it is likewise a specific PHD2 inhibitor that acts via PP2A/B55α activation [17]. VCE-005.1 has demonstrated efficacy in various preclinical models, including neonatal intraventricular hemorrhage [18], hypoxic-ischemic brain injury [19], inflammatory bowel disease [20], and traumatic brain injury (TBI) [21]. In this study, we aimed to investigate the mechanism of action of VCE-005.1 in the B55α/AMPK/SIRT1/HIF pathway and evaluate its efficacy in preclinical models of CLI and DFU. Our findings seek to position VCE-005.1 as a therapeutic candidate for the treatment of DFU and other ischemic vascular diseases Materials and Methods Cell cultures and reagents. Human hair dermal papilla cells (Innoprot, Spain) were immortalized by overexpressing SV40 to generate a human primary transformed cell line (Immortalized Human Hair Dermal Papilla Cells, IHHDPC). These cells, along with the EA.hy926 (ATCC® CRL-2922™) and NIH-3T3-EPO-Luc [22] cell lines were maintained in DMEM (Pan-Biotech, Germany), while the THP-1 cell line (ATCC® TB1-202™) was grown in RPMI 1640 medium (Pan-Biotech). All culture media were supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. Human microvascular endothelial cells (HMEC-1) (ATCC® CRL-3243™) were cultured in MCDB131 medium (Pan-Biotech), supplemented as previously described [10]. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and routine testing was performed to detect mycoplasma contamination and prevent cross-contamination. VCE-005.1 (99,1% purity) was provided by VivaCell Biotechnology España S.L. and dissolved as previously reported [8, 20]. siRNA transfections and Luciferase assays The RNAi assays were conducted using ON-TARGETplus SMARTpools targeting SIRT1 (#L-003540) and B55α (#L-004824), along with a non-specific siRNA control pool (#D-001810) (Dharmacon, Lafayette, CO, USA). A concentration of 10 nM was used in all cases. For luciferase assays, the NIH-3T3-EPO-luc cells (10 4 cells/well) were seeded in 96-well plates. After 24 hours, the cells were pre-treated with the inhibitors for 30 minutes and then exposed to the compound VCE-005.1 for 6 hours. The inhibitors used included: Dorsomorphin (DS) (AMPK inhibitor #S7306, Selleckchem, Houston, TX, USA), EX527 (SIRT1 inhibitor #E7034, Sigma-Aldrich), and CCT018159 (HSP90 inhibitor #7687880, Sigma- Aldrich). After treatment, cells were lysed in 50µL lysis buffer containing 25 mM Trisphosphate (pH 7.8), 8 mM MgCl 2 , 1 mM DTT, 1% Triton X-100, and 7% glycerol. Using a TriStar2 Berthold/LB942 multimode reader (Berthold Technologies, Bad Wildbad, Germany), luciferase activity was measured according to the instructions provided by the luciferase assay kit (Promega, Madison, WI, USA). Western blots After stimulation and washing with phosphate buffered saline (PBS), proteins were harvested using 50 µL of RIPA buffer containing phosphate and protease inhibitors (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.5 mM Na₃VO₄, 50 mM NaF, and 40 µg/µL Protease Inhibitor Cocktail) or with lysis buffer previously described [17]. Proteins (40 µg) were heated at 95°C in Laemmli buffer and separated by electrophoresis on 8%-12% SDS-PAGE gels. Subsequently, proteins were transferred onto nitrocellulose membranes for 30 minutes at 24V. The membranes were then blocked with tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% non-fat dry milk for 1 hour RT. Immunodetection of specific proteins was performed by incubating the membranes with the primary antibody overnight at 4°C (Supplemental Table 1). After washing, the membranes were incubated with a secondary antibody (Supplemental Table 1) for 1 hour and detected using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). All experiments were performed in triplicate. SIRT1 Activity Assay The SIRT1 activity assay was conducted using the fluorometric SIRT1 Activity Assay Kit (#ab156065, Abcam, Cambridge, UK), following the manufacturer's instructions. NAD/NADH Assay The NAD + /NADH assay was performed using the NAD + /NADH assay kit (#ab65348, Abcam), following the manufacturer's instructions. EA.hy926 cells (2 × 10⁶ cells/well) were seeded and stimulated for 4 hours before measuring NAD + /NADH levels. Senescence assay HMEC-1 cells were incubated with different concentrations of VCE-005.1 for one hour and subsequently treated with H 2 O 2 for 4h. This treatment was repeated on days 2 and 5. At day 7, and following the manufacturer's instructions, Senescence-Associated β-Galactosidase (SA-β-gal) staining (#9860, Cell Signaling Technology, Danvers, MA, USA) was performed. Cell viability under high glucose conditions EA.hy926 cells (10⁴ cells/well) were seeded in 96-well plates and pre-treated with increasing concentrations of VCE-005.1 for 1 hour. Subsequently, the cells were exposed to high glucose (HG) concentration (270 mM), while the control group received 5 mM glucose. After 24 hours, 5 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (CT01-5, Sigma-Aldrich) was added at a for 4 hours. The medium was then discarded, DMSO was added, and absorbance was measured using a Microplate Reader (Tecan) at a wavelength of 550 nm. Finally, cell viability was calculated as a percentage in comparison to the control group. Immunocytochemistry analysis Immortalized human hair dermal papilla cells (2 × 10⁴ cells/well) were cultured on glass coverslips placed in 24-well plates. The cells were then exposed to different concentrations of VCE-005.1 for 3h, fixed in a methanol: acetone (1:1) solution at -20°C for 8 minutes and washed with PBS. Next, the cells were blocked with 3% BSA (A6003, Sigma-Aldrich) in PBS for 1 hour and incubated overnight at 4°C with the appropriate primary antibodies (Supplemental Table 1). After washing, the slides were incubated in the dark for 1 hour with the corresponding secondary antibody (Supplemental Table 1). Finally, the samples were mounted using Vectashield Mounting Medium containing 4′,6-diamidino-2-phenylindole (DAPI) (H-1200-10, Vector Laboratories, Burlingame, CA, USA), and images were acquired with a spectral confocal laser-scanning microscope LSM710 (Zeiss, Jena, Germany). THP‑1 cell differentiation and foam cell formation THP-1 cells were cultivated in 24-well plates at a density of 1.5 x 10 6 cells/well, then incubated with 160 nM PMA (Phorbol-12-myristate-13-acetate) (#524400, Sigma-Aldrich) for 24 hours, after which undifferentiated macrophages were removed. Foam cell formation was induced by incubating macrophages for 48 hours with oxLDL (80 µg/ml) (L34358, Thermo Fisher Scientific, MA, USA) or OxLDL plus VCE-005.1. After fixation, a Nile Red solution (N3013, Sigma-Aldrich) was used to stain the cells for 20 minutes. Samples were prepared with Vectashield Mounting Medium with DAPI, and a fluorescence microscope Leica Thunder Imager 3D assay (LEICA, L’Hospitalet de Llobregat, Spain) was used to acquire images. Cell cycle analysis THP-1 cells (5 × 10⁵ cells/well) were plated in 24-well plates and treated with VCE-005.1 for 16 hours. Afterward, the cells were collected using Trypsin-EDTA (0.25%), centrifuged for 5 minutes (3500 rpm, 4°C), and permeabilized in 700 µL of 70% ethanol at 4°C overnight. Following washing, the cells were incubated with PBS containing 1 mg/mL propidium iodide and 5 µL/mL RNase for 3 hours under orbital shaking at room temperature in the dark. Finally, samples were examined via flow cytometry with a BD FACSCanto II flow cytometer (Beckton, Dickinson and Company, New Jersey, USA) to determine PE-A expression. Animals Mice used in the study were sourced from Charles River Laboratories (Barcelona, Spain) and kept with unrestricted access to standard food and water in a controlled environment (temperature 20 ºC (± 2 ºC), 40–50% relative humidity, and a 12-hour light/dark cycle). The experiments were conducted in accordance with European Union regulations and received approval from the Animal Research Ethics Committee of Cordoba University under Project Numbers 01/06/2020/072 and 31/03/2022/057. Mouse aortic ring assay Male C57BL/6 mice were treated daily intraperitoneally (i.p.) either with vehicle (ethanol: cremophor: saline solution 1:1:18) or with VCE-005.1 (30 mg/kg and 60 mg/kg). After seven days of treatment, the thoracic aorta was sectioned into 1 mm aortic rings and cultured in Opti-MEM (51985-034, Thermo Fisher Scientific) supplemented with 1% (v/v) penicillin/streptomycin in a humidified incubator at 37°C and 5% CO 2 and images of the samples were collected at day 10. Critical limb ischemia (CLI) and vascular casting. Male C57BL/6 mice (aged 10–12 weeks) were assigned to two groups, and femoral ligations were performed as previously described [10]. VCE-005.1 (60 mg/kg) was administered i.p. daily throughout the duration of the study. The control group received the vehicle treatment. 10 days later, the mice were anaesthetized and administered 1000 µl of heparin (#9041-08-1, Sigma-Aldrich). They were then perfused and the limb muscles were excised. Following this, the samples were frozen using isopentane cooled in liquid nitrogen. Subsequently, the samples were cut into 5 µm sections at -21°C using a Leica CM1950 Cryostat (Leica Microsystems, Wetzlar, Germany). Vascular casting was performed using Microfil (MV-112 (white), Flow Tech Inc., South Windsor, CT, USA) as indicated in [10]. In vivo wound healing experiments. Male 8-week-old diabetic db/db mice and their normoglycemic heterozygous db/+ controls were divided into 4 groups (4–6 mice/group): db/+ + vehicle group; db/+ + VCE-005.1, db/db + vehicle group and db/db + VCE-005.1. On the day of injury, the mice were anesthetized with isoflurane, and their dorsal hair was removed using an electric razor and depilatory cream. The area was disinfected with alcohol, and two 6 mm wounds were created using a biopsy punch. A rubber splint made of silica gel (internal and external diameters of 8 and 15 mm, respectively) was placed around the wound to prevent closure of the native wound. The splint was secured using SuperGlue and fixed with 4–6 interrupted sutures. 50 µL of VCE-005.1 treatment (cream containing 1% of compound, 5% liquid petrolatum, and 94% solid petrolatum) or vehicle (cream with 100% of solid petrolatum) were applied topically to the wound site every two days using a syringe and a transparent bandage (Tegadem 3M) was used to cover the wound. Digital photographs were taken every two to three days over a period of 10 days. The wound area was measured in pixels using a ruler and a rubber splint, and the analysis was performed using ImageJ 1.32 software ( http://rsbweb.nih.gov/ij/ ). The results were presented as the percentage of wound healing on the days evaluated. Finally, tissue samples were collected, with one wound reserved for histological analysis and the other for mRNA and protein expression studies. Histology and immunohistochemistry Paraffin sections (5 µm) were processed and examined using H&E staining or incubated with Harris Hematoxylin, Acid Fuchsine, Phosphomolybdic acid, and 1% Acetic acid for Masson's Trichrome staining. Skin samples from each mouse were analysed using a semi-quantitative scoring method to assess re-epithelialisation, epithelial thickness index, remodelling and new collagen formation, as described previously [23]. For immunohistochemical (IHC) analysis, tissue sections (5 µm thick) were deparaffinised and subjected to boiling treatment in 1x citrate buffer for 10 minutes, followed by incubation in a solution of 3.3% H 2 O 2 in methanol for 10 minutes. Subsequently, the samples were washed three times with PBS containing 0.1% Tween, treated with a blocking solution (#20773, Merck, Darmstadt, Germany) and left to incubate at 4°C overnight with the corresponding primary antibody (Supplemental Table 1). After that the slides were incubated 1h RT with the appropriate secondary antibody (Supplemental Table 1). Following this, the samples were treated with avidin-biotin-peroxidase reagent (PK-6100, Vector Laboratories) and DAB (3,3'-diaminobenzidine) chromogen (K346811-2, Dako, Santa Clara, CA, USA). The preparations were then counterstained with hematoxylin followed by dehydration and mounted with Eukitt mounting medium. Confocal immunofluorescence. Gastrocnemius muscle samples (5 µm thick) were fixed for 10 minutes at -20°C in a 1:1 methanol-acetone solution, while skin wound samples (5 µm thick) were deparaffinized and prepared for immunofluorescence as described [10]. The samples were then left to incubate overnight at 4°C with the appropriate primary antibody (Supplemental Table 1). The following day, after performing three washes, the secondary antibody was incorporated (Supplemental Table 1). After mounting the slides with Vectashield Mounting Medium with DAPI, images were obtained using a LSM710 laser scanning confocal spectral microscope (Zeiss) with either a 20x/0.8 or 40x oil immersion Plan-Apochromat objective. RNA extraction and qRT-PCR Skin wound tissue and gastrocnemius muscle samples were collected for RNA extraction using the RNeasy Lipid Mini Kit (#74804, Qiagen, Hilden, Germany), in accordance with the manufacturer's instructions. A total of 1 µg of RNA was then reverse transcribed into complementary DNA (cDNA) via the iScript cDNA Synthesis Kit (#1708891, Bio-Rad, Hercules, CA, USA). The resulting cDNA was then subjected to quantitative real-time PCR (qPCR) using the iQ™ SYBR Green Supermix (#1708880, Bio-Rad, Hercules, CA, USA) and a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). PCR efficiency was normalized to the expression of the PPIA gene for each sample, and the relative fold change in gene expression was determined using the -ΔΔCt approach. The primers employed for amplification were synthesized by Eurofins (Luxembourg) and are detailed in the Supplemental Table 2. Olink Proximity Extension Assay (PEA). Plasma samples from different animal groups were submitted to Cobiomic Biosciences SL (Córdoba, Spain), for detection of proteins across the Olink Target 96 Mouse panel (Uppsala, Sweden). Data from Olink PEA were presented as normalized protein expression (NPX) values on a Log2 scale, and their intensity was adjusted using the plate median for each assay to minimize both intra- and inter-assay variation. NPX data was used to detect variations in individual protein levels across samples and helped establish protein signatures by relatively quantifying protein levels. For a list of proteins included in the mouse exploratory panel please visit: https://cobiomicbioscience.com/wp-content/uploads/2024/04/Target-96-Mouse-Exploratory.pdf Quantification and Statistical Analysis All the images were analyzed using the ImageJ software and data were expressed as the mean ± SEM or SD. The Kolmogorov-Smirnov, Shapiro-Wilk or D'Agostino tests and Pearson's test were applied to evaluate the normality of the data. One-way analysis of variance (ANOVA) was then performed, followed by Tukey's or Dunnett's post-hoc tests for parametric comparisons, or the Kruskal-Wallis test for non-parametric analyses. A significance level of p ≤ 0.05 was considered. All statistical analyses were conducted using GraphPad Prism version 9 (GraphPad, San Diego, CA, USA). Results VCE-005.1 stabilizes HIF-1α through a pathway that involves SIRT1 and AMPK and prevents endothelial senescence In previous studies, we demonstrated that the compound VCE-005.1 activates HIF-1α through a PP2A/B55α-dependent pathway, with AMPK signalling also playing a role in is the mechanism of action [17, 21]. AMPK is known for its cardiovascular protective effects, regulating endothelial function, redox homeostasis, and inflammation [23]. Herein we have further examined the effect of VCE-005.1 on AMPK activation and downstream targets such as SIRT1 and eNOS. We found that VCE-005.1 induced AMPK phosphorylation, SIRT1 expression and HIF-1α stabilization in vascular endothelial cells. Dosomorphin (DS), an AMPK inhibitor, blunted VCE-005.1-induced AMPK phosphorylation and SIRT1 expression and attenuated HIF-1α stabilization (Fig. 1 a). Furthermore, knocking down SIRT1 expression (siSIRT1) greatly prevented VCE-005.1-induced HIF-1α activation (Fig. 1 b). The interaction between AMPK and SIRT1 is bidirectional, as AMPK can activate SIRT1 by increasing NAD + levels in cells, thereby promoting its activation. In turn, SIRT1 deacetylates the AMPK upstream kinase LKB1, leading to AMPK phosphorylation and activation [24–26]. We observed that treatment with VCE-005.1 increased both SIRT1 enzymatic activity (Fig. 1 c) and the NAD + /NADH ratio (Fig. 1 d) in EA.hy926 cells. Next, we evaluated the effect of VCE-005.1 on cellular senescence in HMEC-1 cells exposed to oxidative stress induced by H₂O₂. Treatment with VCE-005.1 reduced SA-β-gal + expression, a hallmark of senescence (Fig. 1 e). In addition, VCE-005.1 treatment restored SIRT1 levels, which were diminished by H₂O₂, while downregulating p21, a key senescence marker, which was upregulated in response to oxidative stress (Fig. 1 f). These findings suggest that VCE-005.1 effectively counteracts endothelial senescence. To gain further insight into the mechanism of action of VCE-005.1 on the AMPK/SIRT1/HIF-1α axis, a transactivation luciferase assay was performed in NIH-3T3-EPO-Luc cells treated with VCE-005.1 either in the absence or in the presence of specific inhibitors of AMPK (DS), SIRT1 (EX527), and HSP90 (CCT018159), the latter being a chaperone that interacts with AMPK [27] and associates with HIF-1α to facilitate its nuclear accumulation [28]. All these inhibitors significantly prevented VCE-005-1-induced EPO-Luc transactivation as a surrogated marker of HIF activation. Additionally, we observed that CCT018159 also inhibited HIF-1α and SIRT1 induction in VCE-005-1-treated endothelial cells (Supplemental Fig. 1a and 1b). The impact of VCE-005.1 on eNOS phosphorylation at Ser1177, another key target of AMPK [29] and its relation with B55α was also investigated. Our results demonstrated that VCE-005.1 promoted eNOS phosphorylation, which was significantly reduced in cells knocked down for B55α by siRNA (Supplemental Fig. 1c). These findings highlight the functional interaction between B55α and AMPK in response to VCE-005.1. VCE-005.1 inhibits the formation of foam cells and induces apoptosis in macrophages. Oxidized low-density lipoproteins (ox-LDL) induce an overaccumulation of lipids in macrophages, resulting in the formation of foam cells, a key event in the onset and advancement of atherosclerosis. These cells contribute significantly to the development of atherosclerotic plaques, serving as a major source of the necrotic core [30, 31]. We investigated the effect of VCE-005.1 on ox-LDL-induced foam cell formation and found that it significantly reduced lipid accumulation in ox-LDL-treated macrophages (Fig. 2 a). Mild hypoxia induces macrophage apoptosis, contributing to the regulation of inflammation in diseases such as multiple sclerosis (MS) [32]. Consequently, small hypoxia-mimetic compounds may have similar effects on macrophages. Given their key role in the pathophysiology of atherosclerosis, we investigated whether VCE-005.1 induces macrophage apoptosis. Our results show that VCE-005.1 promotes PARP-1 fragmentation, which coincides with HIF-1α induction (Fig. 2 b). Furthermore, cell cycle analysis revealed a significant increase in the percentage of sub-diploid cells following VCE-005.1 treatment, indicating enhanced apoptosis (Fig. 2 c). Effects of VCE-005.1 on vasculogenesis in vitro and in vivo To assess the influence of VCE-005.1 on vascularization and sprouting, an ex vivo aortic ring assay was performed. The results showed that VCE-005.1 treatment significantly increased the formation of new sprouts, suggesting its ability to promote vascularization in vitro (Fig. 3 a). To further investigate its effects in vivo , a critical limb ischemia (CLI) model was established by performing a double ligation of the left femoral artery. The results showed that VCE-005.1 treatment clearly enhanced arterialization in the limb with the clamped artery without affecting the vascular structure of the control limb (Fig. 3 b). Additionally, the expression mRNA levels of HIF-1α target genes essential for angiogenesis, including hepatocyte growth factor ( Hgf ), erythropoietin ( Epo ), and vascular endothelial growth factor A ( Vegf-A ), were also analysed in the gastrocnemius muscle. The results indicated that the expression levels of these genes were significantly higher in the ligated limb treated with VCE-005.1 compared to the untreated ligated limb. However, this significant induction was not observed in the healthy limb treated with the compound (Fig. 3 c). Caveolin-1 (CAV1), regulated by HIF-1α, plays a essential role in angiogenesis and the vascular response to ischemia [33, 34]. In our study, CAV1 expression was reduced in the ligated limb of untreated CLI mice. However, treatment with VCE-005.1 restored its expression levels, suggesting a protective role in the affected limb (Supplemental Fig. 2a). Moreover, VCE-005.1 stimulated the formation of mature vessels (CD31 + /αSMA + ) and promoted endothelial cell proliferation (CD31 + /Ki67 + ), specifically in the vascular endothelium of the ischemic limb (Fig. 4 a). These findings indicate that VCE-005.1 acts selectively in hypoxic tissues. Moreover, we examined the expression of SIRT1 in the gastrocnemius of CLI mice and we found a decrease in SIRT1 expression in the clamped limb that was reversed in mice treated with VCE-005.1, without affecting the control limb (Fig. 4 b). To evaluate the effects of VCE-005.1 on peripheral biomarkers associated with CLI, the compound was administered i.p. to CLI mice for 7 days and plasmatic biomarkers were analysed using a proximity extension assay. In CLI mice, we observed significant alterations in inflammatory, endothelial, apoptotic, oxidative stress-related, and vascular remodelling pathways (Fig. 5 ). The inflammatory response was characterized by increased levels of tumour necrosis factor-alpha (TNF-α), C-C motif chemokine ligand 2 (CCL2/MCP-1), C-C motif chemokine ligand 3 (CCL3/MIP-1α), and interleukin-10 (IL-10), indicating an imbalance between pro-inflammatory and compensatory anti-inflammatory mechanisms. Endothelial dysfunction and vascular remodelling were evidenced by the upregulation of platelet-derived growth factor subunit B (PDGFB) and WNT1-inducible signalling pathway protein 1 (WISP1), suggesting excessive vascular remodelling and fibrosis. Apoptotic and senescence-associated changes were reflected in the increased expression of FAS cell surface death receptor (FAS), poly [ADP-ribose] polymerase 1 (PARP1), and tumour necrosis factor receptor superfamily member 11B (TNFRSF11B/osteoprotegerin, OPG), suggesting heightened cell death and vascular aging. The oxidative stress response was also altered, as indicated by increased levels of peroxiredoxin-5 (PRDX5), which may represent an attempt to counteract oxidative damage. Neurovascular dysfunction and impaired regenerative capacity were reflected in the upregulation of follistatin (FST) and hepatocyte growth factor (HGF), likely as a response to promote muscle repair and angiogenesis. In VCE-005.1-treated CLI mice, we observed a restoration of inflammatory balance, as levels of TNF-α, CCL2, CCL3, and IL-10 returned to control values. Endothelial stability was improved through modulation of PDGFB and WISP1, suggesting a more controlled vascular remodelling process. Apoptotic and senescence-associated markers FAS, PARP1, and TNFRSF11B were normalized, indicating a protective effect against cell death and vascular aging. The oxidative stress response remained stable with PRDX5 levels maintained, reflecting a sustained antioxidant defence. Neurovascular recovery was enhanced with the further increase of FST and HGF, reinforcing the potential of VCE-005.1 to promote vascular repair and tissue regeneration. These findings suggest that VCE-005.1 mitigates CLI-induced inflammation, apoptosis, and vascular senescence while enhancing endothelial function and neurovascular regeneration, highlighting its potential as a therapeutic strategy in peripheral ischemic conditions. VCE-005.1 protects endothelial cells against glucose-induced cytotoxicity and enhances wound healing in diabetic mice. PAD and DFUs arise from hyperglycemia-induced endothelial dysfunction, promoting atherosclerosis, reduced blood flow, and impaired healing, increasing the risk of complications [35, 36]. To investigate the effect of VCE-005.1 on cell viability under high glucose conditions, EA.hy926 cells were exposed to 270 mM glucose with or without the compound. VCE-005.1 significantly improved cell viability under these conditions, indicating a protective effect against glucose-induced cytotoxicity (Fig. 6 a). To assess the efficacy of VCE-005.1 in diabetic wound healing in vivo , we examined its effects in diabetic db/db mice and their db/+ controls. VCE-005.1 (1% in Vaseline) was applied locally every other day for 10 days, significantly accelerating wound closure compared to the vehicle-treated group (Fig. 6 b and 6 c). This improvement was observed in both heterozygous and homozygous diabetic mice, although untreated diabetic mice exhibited slower wound healing. We also found that topical VCE-005.1 promoted re-epithelialization and increased the skin thickness index 10 days post-injury in both experimental groups. Additionally, tissue remodelling was enhanced, and collagen deposition in the wound area was significantly increased (Fig. 7 a and 7 b). Persistent inflammation is a hallmark of impaired wound healing in diabetes and plays a critical role in tissue repair dysfunction. To assess the impact of VCE-005.1 on immune cell recruitment at the wound site, macrophage and neutrophil infiltration were analysed. VCE-005.1-treated mice exhibited a significant reduction in macrophages, as indicated by F4/80 staining (Fig. 7 c), and neutrophils, as detected by MPO staining (Fig. 7 d), compared to vehicle-treated mice. This effect was observed in both homozygous and heterozygous diabetic animals, suggesting a modulation of the inflammatory response. To further investigate the pro-angiogenic effects of VCE-005.1 in wound healing, vessel density and perimeter were quantified using CD31 + /αSMA + staining, alongside the expression of the endothelial markers CD31 and CD34. VCE-005.1 treatment significantly increased microvessel density and perimeter in the wound area (Fig. 8 a). Moreover, the number of double-stained vessels (CD34 + /CD31 + ) was elevated, indicating the induction of de novo vessel formation. These angiogenic effects were evident in both control and diabetic mice treated with VCE-005.1. The expression mRNA levels of HIF-1α target genes essential for angiogenesis ( HGF, Epo, Vegf-a ) were also analysed in wounded skin tissue. Their expression was significantly reduced in untreated groups compared to VCE-005.1-treated groups (Fig. 8 b), suggesting a role for VCE-005.1 in promoting HIF-1α-mediated angiogenesis. In addition, CAV1 expression, another gene regulated by HIF-1α, was significantly increased following VCE-005.1 treatment in both db/db and db/+ mice. Notably, CAV1 expression was specifically localized to endothelial cells, as confirmed by double staining for CAV1 + /CD31 + . Furthermore, VCE-005.1 treatment not only increased CAV1 protein expression but also significantly upregulated its mRNA levels (Supplemental Fig. 2b, 2c and 2d), reinforcing its role in endothelial function and vascular remodelling. These findings suggest that VCE-005.1 enhances wound healing by reducing excessive inflammation and promoting angiogenesis through HIF-1α signalling in endothelial cells. VCE-005.1 induces the expression of B55α and HIF-1α in endothelial cells in vivo and in vitro As previously demonstrated, our data establish a mechanistic link between B55α and SIRT1 expression and activity in vitro . To further investigate this relationship, we analysed their expression in skin samples following injury. Topical application of VCE-005.1 significantly upregulated B55α and SIRT1 expression in the dermal wound area of both diabetic and control mice compared to untreated controls, suggesting a role in wound healing. Notably, B55α and SIRT1 expression was specifically localized to endothelial cells, as confirmed by double staining for B55α + /CD31 + and SIRT1 + /CD31 + (Fig. 9 a). Furthermore, mRNA levels of both proteins were significantly elevated in the dermis of VCE-005.1-treated db/+ and db/db mice post-injury (Fig. 9 b). These findings suggest that VCE-005.1 promotes endothelial-specific B55α and SIRT1 expression, potentially contributing to its pro-reparative and angiogenic effects during wound healing. Additionally, we found that VCE-005.1 enhanced hair follicle generation, particularly in control mice, suggesting a role in stimulating hair follicle-associated regenerative processes (Fig. 7 a). Hair follicles play a fundamental role in skin tissue repair and wound healing by serving as a reservoir of stem cells and secreting growth factors essential for tissue regeneration. Interestingly, HIF-1α plays a key role in hair follicle regeneration and dermal vascularization [9, 37]. Given that hair dermal papilla cells (HDPCs) are critical for hair follicle regeneration and may contribute to wound healing, we evaluated whether VCE-005.1 could stimulate HIF-1α expression in immortalized HDPC cells (IHDPCs). Our results demonstrated that VCE-005.1 activates both B55α and HIF-1α in a dose-dependent manner, as confirmed by Western blot (Supplemental Fig. 3a) and immunofluorescence (Supplemental Fig. 3b). These findings suggest that VCE-005.1 enhances B55α and HIF-1α expression in endothelial cells and dermal papilla cells, reinforcing its potential to promote vascularization, hair follicle regeneration, and overall skin tissue repair. Discussion PAD and DFUs result from impaired angiogenesis and endothelial dysfunction, which restrict blood flow and compromise tissue healing. Effective revascularization is therefore crucial for restoring circulation and promoting repair. While gene therapies targeting angiogenic factors like VEGF and stem cell-based approaches have shown promise, their clinical application remains limited due to efficacy challenges, safety concerns, and difficulties in achieving sustained effects [38, 39]. These limitations highlight the need for alternative or complementary strategies to enhance vascular regeneration and improve patient outcomes. In this context, our study investigates the potential of VCE-005.1 as a therapeutic agent for endothelial protection and vascular regeneration. Our findings provide evidence that VCE-005.1 exerts significant protective and regenerative effects in preclinical models of critical limb ischemia (CLI) and diabetic wound healing, likely mediated by the activation of the B55α/AMPK/SIRT1/HIF-1α axis, which represent key pathway(s) involved in endothelial function, angiogenesis, prevention of endothelial senescence, and tissue repair. The B55α regulatory subunit of PP2A plays a dual role in mitotic regulation and hypoxia signaling. In mitosis, it ensures cell cycle progression by dephosphorylating key substrates involved in chromosome segregation, nuclear envelope reformation, and anaphase-promoting complex regulation [40, 41]. Simultaneously, B55α interacts with PHD2, modulating its activity through dephosphorylation at Ser125, thereby influencing HIF-1α stability and cellular adaptation to hypoxia [12]. Our results suggest that B55α/PHD2 interaction is one of the primary targets for the vasculogenic activity of VCE-005.1, as no mitotic effects were observed in vitro, despite enhanced vascular endothelial proliferation in vivo. This indicates that vascular regeneration is primarily driven by hypoxia-induced signaling rather than direct mitotic stimulation, with the HIF-1α/VEGF axis likely mediating endothelial expansion and vascular remodeling. Proteomic analysis of the B55α interactome has identified numerous substrates, predominantly proteins involved in mitotic regulation [42]. Notably, AMPK, SIRT1, and PHD2 were not detected in these analyses, despite prior evidence supporting a functional interaction between B55α and PHD2 [12]. This discrepancy may stem from technical limitations of proteomic methods, which often favor the detection of stable or abundant interactions and may overlook transient, low-abundance, or context-specific complexes. Therefore, the absence of these proteins in the interactome should not be interpreted as evidence of a lack of interaction, but rather as a limitation of the methodology. This reinforces the need for complementary approaches to fully elucidate the regulatory network of B55α. In response to B55α activators, such as VCE-005.1 and Etrinabdione, siRNA-mediated B55α knockdown prevents HIF-1α activation, mirroring the effects observed with siRNA targeting SIRT1 and pharmacological inhibition of AMPK and SIRT1 [17, 43]. Notably, other AMPK activators, such as metformin, and SIRT1 activators, such as resveratrol, fail to induce HIF-1α expression. While SIRT1 has been reported to repress HIF-1α activity through deacetylation and inhibition of coactivator recruitment [44], our findings suggest that in the context of VCE-005.1 treatment, SIRT1 may contribute to HIF-1α stabilization in cooperation with AMPK and HSP90. These dual roles highlight the context-dependent nature of the influence of SIRT1 on HIF-1α, suggesting that its effect may vary according to cellular environment, metabolic status, or cofactor availability. Our findings suggest that B55α may function as a central regulatory node in two distinct but interconnected pathways: in one, B55α/PHD2 initiates HIF-1α protein stabilization, while in the other, B55α/AMPK/SIRT1 enhances such stabilization and induces HIF-1α nuclear translocation in coordination with HSP90 [28]. These findings position B55α as a critical modulator of HIF-1α signaling, integrating B55α-mediated inhibition of PHD2 and B55α/AMPK/SIRT1 signalling with significant implications for HIF-1α activation, hypoxia adaptation and vascular remodeling. The precise nature of the interactions between B55α, AMPK, and SIRT1 remains unclear. Direct interaction appears unlikely, as PP2A-mediated AMPK activation by dephosphorylation has not been described. On the contrary, PP2A is generally known to inactivate AMPK by dephosphorylating Thr172, a key activating site [45]. However, the regulation of AMPK is complex and involves multiple phosphorylation sites. Inhibitory phosphorylation can occur at serine residues such as Ser485 on the α1-subunit (or Ser491 on the α2-subunit), which can prevent activation by hindering phosphorylation at Thr172. It is therefore conceivable, though speculative, that under specific conditions, PP2A/B55α might dephosphorylate these inhibitory serine residues, thereby facilitating subsequent activation at Thr172 by upstream kinases such as LKB1, CaMKKβ, or TAK1[46]. Further experimental validation is needed to support this hypothesis. Similarly, no direct regulation of SIRT1 by PP2A has been demonstrated, although indirect regulatory mechanisms may exist. Indeed, proteomic analysis of the B55α interactome has identified numerous substrates, predominantly proteins involved in mitotic regulation [42, 47]. However, AMPK, SIRT1, and PHD2 were not detected, despite prior evidence supporting a direct interaction between B55α and PHD2 [12]. This discrepancy may be due to technical limitations of interactome analysis, which may favor the detection of specific protein subsets, potentially overlooking transient or context-dependent interactions. Further investigation is warranted to determine whether inhibition of B55α interaction with selected substrates as described by Hein et al. [42] can suppress HIF activation and angiogenesis induced by VCE-005.1 or other B55α activators, potentially unveiling new regulatory mechanisms of B55α in hypoxia-driven vascular remodeling. Additionally, we cannot rule out the possibility that VCE-005.1 influences the phosphorylation status of endogenous inhibitors of PP2A/B55α, such as Arspp19, which is regulated by the Greatwall kinase [48]. VCE-005.1 also demonstrated the ability to inhibit foam cell formation and induce apoptosis in macrophages, key contributors to atherosclerotic plaque progression [49]. By reducing lipid accumulation in ox-LDL-treated macrophages, VCE-005.1 may attenuate the formation of necrotic cores within atherosclerotic plaques. Its capacity to induce apoptosis in macrophages, likely through the HIF-1α pathway [50], suggests a potential therapeutic avenue for controlling inflammatory responses in vascular diseases. Additionally, its effect on PARP-1 cleavage supports its role in promoting programmed cell death, reinforcing its anti-atherosclerotic properties. The compound significantly enhanced vascularization and arteriogenesis in both in vitro and in vivo models. The ex vivo aortic ring assay revealed increased new sprout formation, while the CLI model confirmed improved collateral vessel formation following VCE-005.1 treatment. The upregulation of angiogenesis-related genes, including Hgf , Epo , and Vegf-A , further supports the pro-angiogenic activity of VCE-005.1. Notably, its effects were selective for ischemic tissues, as no significant changes were observed in non-ligated limbs, highlighting the ability of the treatment to act in hypoxic tissues. It also promoted the formation of mature blood vessels and enhanced endothelial cell proliferation, demonstrating its efficacy in promoting functional neovascularization. This effect correlated with increased expression of Caveolin-1, a HIF-1α-dependent gene that may play a key role in endothelial function. Chronic hyperglycemia is a major contributor to endothelial dysfunction and impaired wound healing in diabetic patients. Our results show that VCE-005.1 significantly improved endothelial cell viability under high glucose conditions, suggesting a protective effect against glucose-induced cytotoxicity. In a diabetic wound healing model, the compound significantly accelerated wound closure, promoted re-epithelialization, and enhanced tissue remodelling. The observed increase in collagen deposition and microvessel density suggests that VCE-005.1 fosters a pro-healing environment. Additionally, it reduced macrophage and neutrophil infiltration at the wound site, indicating its anti-inflammatory properties. These findings reinforce its therapeutic potential for improving vascular and tissue repair processes in diabetic patients. Our study also highlights the crucial role of B55α and HIF-1α in the mechanism of action of VCE-005.1. Increased expression of B55α and SIRT1 in endothelial cells was observed both in vitro and in vivo following treatment. The selective activation of these proteins in the skin of diabetic mice suggests that VCE-005.1 enhances endothelial-specific responses, promoting tissue regeneration and vascular repair. For instance, the induction of HIF-1α in hair dermal papilla cells suggests a potential role in skin regeneration, which may have broader implications for wound healing and dermatological applications [51, 52]. In summary, our findings establish VCE-005.1 as a promising therapeutic candidate for peripheral artery disease, critical limb ischemia, and diabetic foot ulcers by targeting the B55α/AMPK/SIRT1/HIF-1α axis. The compound promotes endothelial protection, inhibits foam cell formation, enhances angiogenesis and arteriogenesis, protects against hyperglycemia-induced cytotoxicity, and accelerates wound healing. Its selective action in ischemic and damaged tissues underscores its therapeutic potential for vascular and metabolic diseases. Future studies should focus on further characterizing its pharmacokinetic properties and clinical translation to optimize its therapeutic application in ischemic vascular diseases. Conclusions In conclusion, VCE-005.1 exhibits potent regenerative and cytoprotective effects in preclinical models of peripheral artery disease, critical limb ischemia, and diabetic foot ulcers. The compound enhances angiogenesis, promotes endothelial proliferation, reduces inflammation, and protects against hyperglycemia-induced cytotoxicity via activation of the B55α/AMPK/SIRT1/HIF-1α axis. Notably, VCE-005.1 selectively targets hypoxic and ischemic tissues, minimizing effects in normoxic regions. Systemic biomarker profiling further supports its therapeutic action, showing restoration of inflammatory balance, attenuation of endothelial dysfunction, normalization of apoptotic and senescence markers, and enhancement of neurovascular recovery. These findings underscore its ability to modulate key pathogenic mechanisms, including vascular remodeling, oxidative stress, and immune dysregulation, associated with CLI. By targeting both cellular and systemic pathways involved in vascular damage and repair, VCE-005.1 emerges as a promising candidate for the treatment of ischemic and metabolic vascular diseases. Further studies are warranted to optimize its clinical translation and explore its long-term efficacy and safety. Abbreviations AMPK AMP-activated protein kinase B55α PP2A Protein phosphatase 2A-B55α subunit (PPP2R2A) BAH Betulinic acid hydroxamate BSA Bovine serum albumin CAV1 Caveolin 1 CCL2/MCP-1 C-C motif chemokine ligand 2 CCL3/MIP-1α C-C motif chemokine ligand 3 cDNA Complementary DNA CLI Critical limb ischemia DFU Diabetic foot ulcers DS Dorsomorphin ECs Endothelial cells EGF Epidermal growth factor eNOS Endothelial nitric oxide synthase EPO Erythropoietin FAS FAS cell surface death receptor FBS Fetal bovine serum FST Follistatin H&E Hematoxylin and Eosin HDPCs Hair dermal papilla cells HG High glucose Hgf Hepatocyte growth factor HIF-1α Hypoxia-inducible factor 1-alpha HMEC-1 Human microvascular endothelial cells i.p. intraperitoneally IF Immunofluorescence IHC Immunohistochemical IHHDPC Immortalized Human Hair Dermal Papilla Cells MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NAD + Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide hydrogen NPX Normalized Protein Expression Ox-LDL Oxidized low-density lipoprotein PAD Peripheral artery disease PARP1 Poly(ADP-ribose) polymerase-1 PBS Phosphate buffered saline PEA Proximity extension assay PRDX5 Peroxiredoxin-5 PHD2 Prolyl hydroxylase 2 PHDs Prolyl hydroxylases PMA Phorbol-12-myristate-13-acetate qPCR Quantitative real-time PCR ROS Reactive oxygen species SD Standard deviation SIRT1 Sirtuin 1 TBI Traumatic brain injury TBS Tris-buffered saline TNFRSF11B Osteoprotegerin, OPG. Tumour necrosis factor receptor superfamily member 11B TNFα Tumour necrosis factor-alpha VCE-004.8 Etrinabdione or EHP-101, [(1′R,6′R)-3-(benzylamine)- 6-hydroxy-3′ -methyl -4-pentyl -6′ - (prop-1-en-2-yl ) [1,1′bi(cyclohexane)]-2′,3,6-triene-2,5-dione) VCE-005.1 BAH or 3β-hydroxylup-20(29)-en-28-oic acid hydroxamate VEGF vascular endothelial growth factor Vegfa Vascular endothelial growth factor A WISP1 WNT1-inducible signalling pathway protein 1 Declarations Ethics Declarations All experimental protocols followed the guidelines of animal care set by the EU guidelines 86/609/EEC, the Ethic Committee on Animal Experimentation at the University of Córdoba (Spain) and the Andalusian Regional Committee for Animal Experimentation approved all the procedures described in this study (31/03/2022/057). Consent for publication Not applicable Availability of data and materials Competing interest None Funding This study was supported by grant CPP2021-008557/AEI/10.13039/501100011033/ Unión Europea NextGenerationEU/PRTR, grant PID2023-148340OB-I00 (Agencia Estatal de Investigación, Spain; co-funded with EU funds from FEDER Program), and CNS2022-135922/ Unión Europea NextGenerationEU/Plan de Recuperación, Tranformación y Resilencia, Agencia Estatal de Investigación. Authors contributions ILC, MEP, JJFS, IM, and AGM performed the experiments and data analysis. AGM and EM designed the overall study, managed and supervised the study. ILC and EM wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements We acknowledge the Advanced Optical Microscopy Unit and the Animal Experimentation Facilities of the IMIBIC. References Bonaca MP, Hamburg NM, Creager MA (2021) Contemporary Medical Management of Peripheral Artery Disease. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6319136","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434732492,"identity":"1ee475f8-74ad-468e-87bf-c9bc3e436245","order_by":0,"name":"Isabel Lastres-Cubillo","email":"","orcid":"","institution":"Maimonides Biomedical Research Institute of Córdoba (IMIBIC)","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Lastres-Cubillo","suffix":""},{"id":434732493,"identity":"f26c82cb-540c-4c5b-a038-7e55480388c2","order_by":1,"name":"María E. Prados","email":"","orcid":"","institution":"VivaCell Biotechnology España","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"E.","lastName":"Prados","suffix":""},{"id":434732494,"identity":"be465d76-c4dd-4dec-86c5-e2debb4a3352","order_by":2,"name":"Juan J. Ferres-Serrano","email":"","orcid":"","institution":"Maimonides Biomedical Research Institute of Córdoba (IMIBIC)","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"J.","lastName":"Ferres-Serrano","suffix":""},{"id":434732495,"identity":"ab610f4e-e556-48b7-9bf6-cf36e9299071","order_by":3,"name":"Ignacio Muñoz","email":"","orcid":"","institution":"Maimonides Biomedical Research Institute of Córdoba (IMIBIC)","correspondingAuthor":false,"prefix":"","firstName":"Ignacio","middleName":"","lastName":"Muñoz","suffix":""},{"id":434732496,"identity":"bc03dfa6-7cb4-48a6-bb35-8723398dd9a7","order_by":4,"name":"Adela García-Martín","email":"","orcid":"","institution":"Maimonides Biomedical Research Institute of Córdoba (IMIBIC)","correspondingAuthor":false,"prefix":"","firstName":"Adela","middleName":"","lastName":"García-Martín","suffix":""},{"id":434732497,"identity":"3e91a211-7911-4692-8363-94ed91025077","order_by":5,"name":"Eduardo Muñoz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYBAC9mbGBgbGBgsGfqK18BxmBmmRYJBsIFrLAXaIFoMDRGthZ2zdzLtDQs74RvLDBwwVdURoYWZsu817RsLY7EaasQHDmcOEtdiDtbRJJG67kcMmwdhGhPN4oFrqN88AaflHtMPaJBIMJEBaGpiJ03Jz7hkJwxlnnhkbJBwjwi88/Mef3Xi7w0aevx0YYh9qiHAYKkggVcMoGAWjYBSMAuwAAIgTNMS6cq8eAAAAAElFTkSuQmCC","orcid":"","institution":"Maimonides Biomedical Research Institute of Córdoba (IMIBIC)","correspondingAuthor":true,"prefix":"","firstName":"Eduardo","middleName":"","lastName":"Muñoz","suffix":""}],"badges":[],"createdAt":"2025-03-27 09:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6319136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6319136/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79580717,"identity":"8add640c-c406-48cd-b85c-c33ef546fbdf","added_by":"auto","created_at":"2025-03-31 11:50:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":479363,"visible":true,"origin":"","legend":"\u003cp\u003eVCE-005.1 activates AMPK/SIRT1 pathways and prevents cellular senescence in endothelial cells. \u003cstrong\u003e(a)\u003c/strong\u003e EA.hy926 cells were pre-stimulated with or without DS for 30 minutes and then treated with VCE.005.1 for 3 hours. The expression levels of pAMPK, AMPKt, SIRT1 and Actin were evaluated using Western blot technique (n=3). \u003cstrong\u003e(b)\u003c/strong\u003e EA.hy926 cells were subjected to transfection with siSIRT1 or scrambled control siRNAs, followed by exposure to VCE-005.1 for a duration of 3h. The expression of SIRT1 and HIF-1α was analyzed by Western Blot (n=3). \u003cstrong\u003e(c)\u003c/strong\u003e SIRT1 induction by VCE-005.1 was detected by a fluorometric assay, utilizing SRT1720 as the positive control. Data represent the mean ±SD (n=3). \u003cstrong\u003e(d)\u003c/strong\u003e NAD\u003csup\u003e+\u003c/sup\u003e/NADH ratio was measured using the EA.hy926 cell line treated with different concentrations of VCE-005.1 for 3 hours. Data represent the mean ±SD (n=3-4); ***p\u0026lt;0.001, ****p\u0026lt;0.0001. \u003cstrong\u003e(e)\u003c/strong\u003e In HMEC-1 cells, VCE-005.1 was found to prevent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cellular senescence, as evidenced by the reduction in SA-β-gal staining. \u003cstrong\u003e(f)\u003c/strong\u003e SIRT1, p21, and Actin protein levels were analyzed by Western blot in HMEC-1 cells under H₂O₂-induced stress, with or without VCE-005.1 treatment (n=3).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/29b94bf8573b9de0ca611a48.png"},{"id":79579679,"identity":"c2ebfe2c-ff9d-437f-a5c9-8515940431fb","added_by":"auto","created_at":"2025-03-31 11:42:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":409681,"visible":true,"origin":"","legend":"\u003cp\u003eVCE-005.1 alleviates ox-LDL damage and induces apoptosis in THP-1 cells.\u003cstrong\u003e (a) \u003c/strong\u003eFoam cell formation was induced by incubating macrophages for 48 hours with oxLDL (80 µg/ml) or oxLDL + VCE-005.1. Lipid droplets were then measured by Nile Red staining. Scale bars equivalent to 50µm. The data passed the normality test performed by the Shapiro-Wilk test. P values were calculated using one-way ANOVA followed by Dunnett's post hoc multiple comparisons to compare between groups. Data are presented as mean ± SD (n = 4-6). P values indicated in the panels are significant as ****p\u0026lt;0.0001. \u003cstrong\u003e(b)\u003c/strong\u003e THP-1 cells were exposed to varying concentrations of VCE-005.1 for 3h, using Etoposide as a positive control. HIF1 and PARP1 expression levels were measured by Western Blot (n=3). \u003cstrong\u003e(c)\u003c/strong\u003e THP-1 cells were treated with or without VCE-005.1 for 16 hours and stained with propidium iodide (PI). The data represent the distribution of cells across different phases of the cell cycle, including apoptosis, G0/G1, S, and G2/M phases. Flow cytometry analysis was performed, collecting 5,000 events per sample to determine PE-A expression (n=3).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/3c7ff960718378865acbfddf.png"},{"id":79581648,"identity":"f8137fab-14a7-497e-bbac-1648afa13bac","added_by":"auto","created_at":"2025-03-31 11:58:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4217199,"visible":true,"origin":"","legend":"\u003cp\u003eVCE-005.1 induces angiogenesis \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003e(a)\u003c/strong\u003eRepresentative images of the aortic ring assay in mice treated intraperitoneally with doses of 30 mg/kg or 60 mg/kg of VCE-005.1. The scales are indicated in the images. \u003cstrong\u003e(b)\u003c/strong\u003e Representative images of vascular casting after 10 days of femoral artery ligation. Black arrows indicate collateral artery growth and scale bar represents 5 mm. \u003cstrong\u003e(c) \u003c/strong\u003eThe mRNA expression of \u003cem\u003eHgf, Epo \u003c/em\u003eand\u003cem\u003e Vegf-a \u003c/em\u003ewere measured in the gastrocnemius muscle after 10 days of ischemia.\u003cstrong\u003e \u003c/strong\u003eThe Shapiro-Wilk test was used to verify the normality of the data. For statistical analysis, one-way ANOVA was applied, followed by Tukey’s post hoc test to evaluate differences between groups (n = 4 animals per group). The results are expressed as mean values ± SEM. Statistical significance is indicated in the figure, with *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/02168468c158a00e592bfa21.png"},{"id":79579689,"identity":"4c3abafe-dcb5-409e-bf7d-841aab6b0929","added_by":"auto","created_at":"2025-03-31 11:42:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5100624,"visible":true,"origin":"","legend":"\u003cp\u003eVCE-005.1 promotes vessel formation, endothelial cell proliferation, and SIRT1 expression in CLI mice.\u003cstrong\u003e\u0026nbsp; (a) \u003c/strong\u003eDouble immunofluorescence confocal staining of α-SMA (green)/CD31 (red) and CD31(green)/Ki67(red) in gastrocnemius muscles 10 days after ischemia (Scale bars=50 μm). Quantification of the number of vessel peer area and the number of CD31\u003csup\u003e+\u003c/sup\u003e/Ki67\u003csup\u003e+\u003c/sup\u003e positive cells. Since the data did not pass the normality test, P values were calculated using a nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test. The results are presented as mean ± SEM. Statistical significance is indicated in the panel, with ***p \u0026lt; 0.001 and ****p \u0026lt; 0.0001. \u003cstrong\u003e(b)\u003c/strong\u003e Confocal double immunofluorescence staining of SIRT1 (green) and CD31 (red) in gastrocnemius muscles after 10 days, along with quantification of SIRT1 expression. Scale bars represent 50 μm. The data did not pass the normality test, and significance was determined using a non-parametric Kruskal-Wallis test. Values are mean ± SEM (n = 3–4). P values are indicated in the panels, with significance denoted as *p \u0026lt; 0.1.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/408a46bc19b79ca66e321014.png"},{"id":79580718,"identity":"d1d4e195-7127-4e2c-a75d-06bb548c5ac6","added_by":"auto","created_at":"2025-03-31 11:50:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":802641,"visible":true,"origin":"","legend":"\u003cp\u003eSignificant Proteins expression Olink CLI model with VCE-005.1 (60mg/Kg) at 7 days. The Target 96 Mouse Exploratory Panels were used to measure various plasma protein concentrations in CLI mice. Normality of the data was confirmed using the Shapiro-Wilk test. Statistical analysis was performed via one-way ANOVA, followed by Dunnett’s or \u003ca href=\"https://www.graphpad.com/guides/prism/latest/statistics/stat_holms_multiple_comparison_test.htm\"\u003eHolm-Šídák\u003c/a\u003e post hoc test to compare differences among the groups (n = 3-4 animals per group). The results are presented as mean values ± SEM. Statistical significance is indicated in panel, with *p \u0026lt; 0.05, **p \u0026lt; 0.01 and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/65697a37944c60638de49a4f.png"},{"id":79580722,"identity":"cfd5818d-1623-4b20-9907-eb78622636af","added_by":"auto","created_at":"2025-03-31 11:50:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3008822,"visible":true,"origin":"","legend":"\u003cp\u003eVCE-005.1 protects endothelial cells under high glucose conditions and promotes wound healing \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003e(a)\u003c/strong\u003e EA.hy926 cells were treated with VCE-005.1 under high glucose conditions, and cell viability was measured using an MTT assay. The data were assessed for normality using the Shapiro-Wilk test. P-values were determined by one-way ANOVA, followed by Tukey's post hoc test for multiple comparisons between groups. Results are presented as the mean ± SD (n = 3). Statistical significance is indicated in the panels, with **p \u0026lt; 0.01 and ***p \u0026lt; 0.001. \u003cstrong\u003e(b)\u003c/strong\u003e Visual analysis of the wounds on each study day. \u003cstrong\u003e(c)\u003c/strong\u003eQuantitative analysis of wound area and percentage closure was performed on days 0, 3, 6, 8, and 10. \u0026nbsp;The Shapiro-Wilk test was used to assess the normality of the data. To compare differences between groups (n = 4-6 animals per group), statistical analysis was carried out using one-way ANOVA followed by Tukey's post hoc test. The results are presented as the mean ± SEM. Statistical significance is indicated in the panels, with *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/317cb47186342872e191e280.png"},{"id":79579699,"identity":"180c4a5e-24c5-46d2-8c5d-1a99405586c3","added_by":"auto","created_at":"2025-03-31 11:42:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6844109,"visible":true,"origin":"","legend":"\u003cp\u003eVCE-005.1 induced re-epithelialization and preventedinflammation. \u003cstrong\u003e(a) \u003c/strong\u003eTissue samples were stained with H\u0026amp;E or Masson’s Trichrome on day 10. The scales are indicated in the images (100 μm). \u003cstrong\u003e(b)\u003c/strong\u003e Re-epithelialization, ETI, Remodeling, and Collagen were quantified based on H\u0026amp;E or Masson staining. The criteria to evaluate the histological score of wound healing was as follows: re-epithelialization (0: None (0%), 1: Partial (1-95%), 2: Complete (95-100%)); ETI (0:Hypoplasia (\u0026lt;95%), 1:Hipertrofia (\u0026gt;105%), 2 healed wound (95-105%)), Remodeling (0: not remodeled, 1: Partial, 2: Complete), Collagen (0: Absent, 1: Minimal-GT (Granulation tissue), 2: Mild-GT, 3: Moderate-GT, 4: Marked-GT). The results are presented as mean values ± SEM (n=4 animals per group). Statistical significance is indicated in panel, with *p \u0026lt; 0.05 and **p \u0026lt; 0.01 (one-way ANOVA followed Dunnett's test). \u003cstrong\u003e(c)\u003c/strong\u003e VCE-005.1\u003cstrong\u003e \u003c/strong\u003eprevented the accumulation of macrophages (F4/80). Scale bar represents 100 μm. Normality of the data was confirmed using the D’Angostino \u0026amp; Pearson test. Statistical analysis was performed via one-way ANOVA, followed by Tukey’s post hoc test to compare differences among the groups (n = 4-6 animals per group). The results are presented as mean values ± SEM. Statistical significance is indicated in panel, with ***p \u0026lt; 0.001 and ****p \u0026lt; 0.0001. \u003cstrong\u003e(d)\u003c/strong\u003e VCE-005.1 reduced neutrophil infiltration (MPO). The scale bar represents 100 μm. Data normality was assessed using the Shapiro-Wilk test. One-way ANOVA, followed by Tukey's post hoc test, was employed for statistical analysis to compare differences between groups (n = 4-6 animals per group). The results are shown as mean ± SEM, with statistical significance indicated in the panel, where ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/b0751c643c17a3d8c2788050.png"},{"id":79580720,"identity":"87cdd6d7-0dd3-4507-bc4c-b7a3a3954ef6","added_by":"auto","created_at":"2025-03-31 11:50:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3934125,"visible":true,"origin":"","legend":"\u003cp\u003eTopical treatment of VCE-005.1 in mice with or without diabetes promoted angiogenesis \u003cem\u003ein vivo. \u003c/em\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Double immunofluorescence confocal staining of CD31 (red)/αSMA (green) and CD34 (green)/CD31 (red) in tissue samples was used to calculate the number of CD31\u003csup\u003e+\u003c/sup\u003e/αSMA\u003csup\u003e+\u003c/sup\u003e cells (vessels formation) and CD34\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e (mature endothelial vascular cells) (scale bars=50 μm)\u0026nbsp; and its quantifications. \u0026nbsp;The measurements for number of vessels and CD31 (Fluorescence intensity) were normally distributed, and statistical analysis was conducted using one-way ANOVA, followed by Tukey’s post hoc test to assess differences between groups (n = 4 animals per group). The data that did not follow a normal distribution were analyzed using a non-parametric approach, followed by a Kruskal-Wallis test to determine statistical significance. Values are mean ± SEM (n = 3–4). P values indicated in panels, significant as **p \u0026lt; 0.01, ***p \u0026lt; 0.001 and ****p \u0026lt; 0.001. \u003cstrong\u003e(b) \u003c/strong\u003emRNA expression levels of \u003cem\u003eEpo\u003c/em\u003e, \u003cem\u003eHgf,\u003c/em\u003e and \u003cem\u003eVegf-a\u003c/em\u003e in the skin after 10 days of wounding. Data normality was assessed using the Shapiro-Wilk test, with statistical comparisons made using one-way ANOVA and Tukey’s post hoc test (n = 4-6 animals per group). Results are presented as mean ± SEM. Statistical significance is marked in the panels, with *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/12c1711b60f8d43d2056c801.png"},{"id":79579698,"identity":"693fac05-f96c-434b-97a2-71896ea6c2b4","added_by":"auto","created_at":"2025-03-31 11:42:25","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3722492,"visible":true,"origin":"","legend":"\u003cp\u003eVCE-005.1 induces B55α and SIRT1 in Wound healing.\u003cstrong\u003e (a) \u003c/strong\u003eRepresentative double immunofluorescence confocal staining of B55α (red)/CD31 (green) and SIRT1 (green)/CD31 (red) in wound healing, along with their quantifications. Scale bars equivalent to 50 μm for confocal images. The quantifications for B55α did not pass the normality test, and significance was determined using non-parametric methods, followed by a Kruskal–Wallis test. For SIRT1, the quantifications passed the normality test, and statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test to compare differences among the groups. Results are presented as mean values ± SEM. Statistical significance is indicated in panel, with *p \u0026lt; 0.05, ***p \u0026lt; 0.001 and ****p \u0026lt; 0.0001. \u003cstrong\u003e(b)\u003c/strong\u003e Expression of \u003cem\u003eSIRT1 \u003c/em\u003eand \u003cem\u003eB55α\u003c/em\u003e mRNA in mice skin at 10 days after wounding.Normality of the data was confirmed using the Shapiro-Wilk test. Statistical analysis was performed via one-way ANOVA, followed by Tukey’s post hoc test to compare differences among the groups (n = 3-6 animals per group). The results are presented as mean values ± SEM. Statistical significance is indicated in panel, with *p \u0026lt; 0.05 and **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/95e1cdd4f3a9d179804a48a9.png"},{"id":79986635,"identity":"2855ffa8-b7f4-4727-969d-b387a995a50f","added_by":"auto","created_at":"2025-04-06 08:16:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25505863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/d1f27aa7-8be2-46f4-896e-829d234d341d.pdf"},{"id":79579686,"identity":"5190315b-13af-4921-831b-0a30692513f6","added_by":"auto","created_at":"2025-03-31 11:42:25","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1801555,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalinformation.250325.docx","url":"https://assets-eu.researchsquare.com/files/rs-6319136/v1/210144cc55e4da135b8872ab.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"B55α Orchestrates AMPK/SIRT1/HIF-1α Signaling: VCE-005.1 as a Tissue-Selective Therapeutic Strategy for Ischemic Vascular Diseases","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeripheral artery disease (PAD) is a chronic vascular disorder primarily caused by atherosclerosis, leading to arterial narrowing or occlusion, particularly in the lower extremities. This restriction in blood flow results in ischemia, manifesting as intermittent claudication, pain, and fatigue during physical activity [1]. If left untreated, PAD can progress to critical limb ischemia (CLI), a severe condition associated with non-healing ulcers, gangrene, and a significantly increased risk of amputation [2]. Globally, PAD affects over 200\u0026nbsp;million individuals, with its prevalence rising due to common risk factors such as smoking, diabetes, hypertension, and dyslipidemia [3].\u003c/p\u003e \u003cp\u003eDiabetes further exacerbates PAD, predisposing individuals to complications such as diabetic foot ulcers (DFUs). These ulcers develop in approximately 15\u0026ndash;25% of diabetic patients during their lifetime and are frequently associated with vascular insufficiency and neuropathy. Notably, nearly 50% of DFU cases co-occur with PAD, leading to impaired wound healing, recurrent infections, and increased amputation rates [4]. The interplay between reduced blood flow, endothelial dysfunction, and impaired tissue repair in PAD and DFU results in chronic peripheral tissue hypoxia, posing a major clinical challenge [4, 5]. These conditions collectively contribute to significant morbidity, mortality, and healthcare costs, highlighting the urgent need for innovative therapeutic strategies targeting endothelial dysfunction and impaired vasculogenesis.\u003c/p\u003e \u003cp\u003eIn PAD and DFU, ischemia and hypoxia trigger compensatory pro-angiogenic mechanisms, notably via the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and growth factors such as vascular endothelial growth factor (VEGF). However, chronic endothelial dysfunction-driven by oxidative stress, inflammation, and impaired endothelial cell signaling-compromises the formation of functional blood vessels. Despite elevated VEGF expression, the damaged vascular microenvironment results in defective or insufficient angiogenesis, failing to restore adequate perfusion or support tissue regeneration [2, 6]. This underscores endothelial dysfunction as a critical barrier to effective angiogenic therapies in PAD and DFU.\u003c/p\u003e \u003cp\u003eHIF-1α is a key transcriptional regulator of the cellular response to hypoxia, orchestrating the expression of pro-angiogenic factors such as VEGF, hepatocyte growth factors (HGF), and erythropoietin (EPO). Under hypoxic conditions, prolyl hydroxylases (PHDs) are inhibited, allowing HIF-1α stabilization and activation of genes essential for angiogenesis and vascular remodeling [7]. Hypoxic preconditioning, achieved through controlled exposure to low oxygen levels or hypoxia-mimetic compounds, has demonstrated potential benefits in vascular diseases by enhancing HIF-dependent pathways, improving tissue oxygenation, and promoting vascular repair [8, 9]. While this strategy holds promise for ischemic conditions like PAD and CLI [10, 11], its therapeutic potential in DFU remains largely unexplored.\u003c/p\u003e \u003cp\u003eRecent research suggests that the regulatory subunit B55α (PPP2R2A) of protein phosphatase 2A (PP2A) could be a novel pharmacological target in vascular diseases. The B55α subunit dephosphorylates PHD2 at Ser125, reducing its activity and subsequently enhancing HIF-1α accumulation and signaling [12]. Additionally, B55α stabilizes endothelial cells (ECs), protecting them against oxidative stress and apoptosis during vascular remodeling [13]. This mechanism not only preserves endothelial integrity but also fosters angiogenesis, supporting new blood vessel formation. On the other hand, SIRT1, a NAD\u003csup\u003e+\u003c/sup\u003e-dependent deacetylase, is a pivotal regulator of vascular function, maintaining endothelial homeostasis and promoting vasodilation, angiogenesis, and tissue regeneration[14]. SIRT1 activity is tightly linked to AMPK, a master regulator of cellular metabolism and vascular function [15]. Dysregulation of the AMPK-SIRT1 axis contributes to endothelial dysfunction and impaired vascular repair in PAD and DFU [16], further supporting the therapeutic potential of AMPK-SIRT1-targeting compounds.\u003c/p\u003e \u003cp\u003eGiven its role in vascular homeostasis, PP2A/B55α activators such as VCE-005.1 and VCE-004.8 (Etrinabdione) emerge as promising candidates for therapeutic intervention in PAD, CLI, and DFU. While Etrinabdione is already in phase IIa clinical trial for PAD patients (clinicaltrials.gov: NCT06774040), VCE-005.1, a betulinic hydroxamate, it is likewise a specific PHD2 inhibitor that acts via PP2A/B55α activation [17]. VCE-005.1 has demonstrated efficacy in various preclinical models, including neonatal intraventricular hemorrhage [18], hypoxic-ischemic brain injury [19], inflammatory bowel disease [20], and traumatic brain injury (TBI) [21].\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate the mechanism of action of VCE-005.1 in the B55α/AMPK/SIRT1/HIF pathway and evaluate its efficacy in preclinical models of CLI and DFU. Our findings seek to position VCE-005.1 as a therapeutic candidate for the treatment of DFU and other ischemic vascular diseases\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eCell cultures and reagents.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHuman hair dermal papilla cells (Innoprot, Spain) were immortalized by overexpressing SV40 to generate a human primary transformed cell line (Immortalized Human Hair Dermal Papilla Cells, IHHDPC). These cells, along with the EA.hy926 (ATCC\u0026reg; CRL-2922\u0026trade;) and NIH-3T3-EPO-Luc [22] cell lines were maintained in DMEM (Pan-Biotech, Germany), while the THP-1 cell line (ATCC\u0026reg; TB1-202\u0026trade;) was grown in RPMI 1640 medium (Pan-Biotech). All culture media were supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. Human microvascular endothelial cells (HMEC-1) (ATCC\u0026reg; CRL-3243\u0026trade;) were cultured in MCDB131 medium (Pan-Biotech), supplemented as previously described [10]. All cell lines were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ and routine testing was performed to detect mycoplasma contamination and prevent cross-contamination. VCE-005.1 (99,1% purity) was provided by VivaCell Biotechnology Espa\u0026ntilde;a S.L. and dissolved as previously reported [8, 20].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003esiRNA transfections and Luciferase assays\u003c/h2\u003e \u003cp\u003eThe RNAi assays were conducted using ON-TARGETplus SMARTpools targeting SIRT1 (#L-003540) and B55α (#L-004824), along with a non-specific siRNA control pool (#D-001810) (Dharmacon, Lafayette, CO, USA). A concentration of 10 nM was used in all cases. For luciferase assays, the NIH-3T3-EPO-luc cells (10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in 96-well plates. After 24 hours, the cells were pre-treated with the inhibitors for 30 minutes and then exposed to the compound VCE-005.1 for 6 hours. The inhibitors used included: Dorsomorphin (DS) (AMPK inhibitor #S7306, Selleckchem, Houston, TX, USA), EX527 (SIRT1 inhibitor #E7034, Sigma-Aldrich), and CCT018159 (HSP90 inhibitor #7687880, Sigma- Aldrich). After treatment, cells were lysed in 50\u0026micro;L lysis buffer containing 25 mM Trisphosphate (pH 7.8), 8 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, 1% Triton X-100, and 7% glycerol. Using a TriStar2 Berthold/LB942 multimode reader (Berthold Technologies, Bad Wildbad, Germany), luciferase activity was measured according to the instructions provided by the luciferase assay kit (Promega, Madison, WI, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blots\u003c/h3\u003e\n\u003cp\u003eAfter stimulation and washing with phosphate buffered saline (PBS), proteins were harvested using 50 \u0026micro;L of RIPA buffer containing phosphate and protease inhibitors (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.5 mM Na₃VO₄, 50 mM NaF, and 40 \u0026micro;g/\u0026micro;L Protease Inhibitor Cocktail) or with lysis buffer previously described [17]. Proteins (40 \u0026micro;g) were heated at 95\u0026deg;C in Laemmli buffer and separated by electrophoresis on 8%-12% SDS-PAGE gels. Subsequently, proteins were transferred onto nitrocellulose membranes for 30 minutes at 24V. The membranes were then blocked with tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% non-fat dry milk for 1 hour RT. Immunodetection of specific proteins was performed by incubating the membranes with the primary antibody overnight at 4\u0026deg;C (Supplemental Table\u0026nbsp;1). After washing, the membranes were incubated with a secondary antibody (Supplemental Table\u0026nbsp;1) for 1 hour and detected using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). All experiments were performed in triplicate.\u003c/p\u003e\n\u003ch3\u003eSIRT1 Activity Assay\u003c/h3\u003e\n\u003cp\u003eThe SIRT1 activity assay was conducted using the fluorometric SIRT1 Activity Assay Kit (#ab156065, Abcam, Cambridge, UK), following the manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003eNAD/NADH Assay\u003c/h3\u003e\n\u003cp\u003eThe NAD\u003csup\u003e+\u003c/sup\u003e/NADH assay was performed using the NAD\u003csup\u003e+\u003c/sup\u003e/NADH assay kit (#ab65348, Abcam), following the manufacturer's instructions. EA.hy926 cells (2 \u0026times; 10⁶ cells/well) were seeded and stimulated for 4 hours before measuring NAD\u003csup\u003e+\u003c/sup\u003e/NADH levels.\u003c/p\u003e\n\u003ch3\u003eSenescence assay\u003c/h3\u003e\n\u003cp\u003eHMEC-1 cells were incubated with different concentrations of VCE-005.1 for one hour and subsequently treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4h. This treatment was repeated on days 2 and 5. At day 7, and following the manufacturer's instructions, Senescence-Associated β-Galactosidase (SA-β-gal) staining (#9860, Cell Signaling Technology, Danvers, MA, USA) was performed.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell viability under high glucose conditions\u003c/h2\u003e \u003cp\u003eEA.hy926 cells (10⁴ cells/well) were seeded in 96-well plates and pre-treated with increasing concentrations of VCE-005.1 for 1 hour. Subsequently, the cells were exposed to high glucose (HG) concentration (270 mM), while the control group received 5 mM glucose. After 24 hours, 5 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (CT01-5, Sigma-Aldrich) was added at a for 4 hours. The medium was then discarded, DMSO was added, and absorbance was measured using a Microplate Reader (Tecan) at a wavelength of 550 nm. Finally, cell viability was calculated as a percentage in comparison to the control group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunocytochemistry analysis\u003c/h3\u003e\n\u003cp\u003eImmortalized human hair dermal papilla cells (2 \u0026times; 10⁴ cells/well) were cultured on glass coverslips placed in 24-well plates. The cells were then exposed to different concentrations of VCE-005.1 for 3h, fixed in a methanol: acetone (1:1) solution at -20\u0026deg;C for 8 minutes and washed with PBS. Next, the cells were blocked with 3% BSA (A6003, Sigma-Aldrich) in PBS for 1 hour and incubated overnight at 4\u0026deg;C with the appropriate primary antibodies (Supplemental Table\u0026nbsp;1). After washing, the slides were incubated in the dark for 1 hour with the corresponding secondary antibody (Supplemental Table\u0026nbsp;1). Finally, the samples were mounted using Vectashield Mounting Medium containing 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) (H-1200-10, Vector Laboratories, Burlingame, CA, USA), and images were acquired with a spectral confocal laser-scanning microscope LSM710 (Zeiss, Jena, Germany).\u003c/p\u003e\n\u003ch3\u003eTHP‑1 cell differentiation and foam cell formation\u003c/h3\u003e\n\u003cp\u003eTHP-1 cells were cultivated in 24-well plates at a density of 1.5 x 10\u003csup\u003e6\u003c/sup\u003e cells/well, then incubated with 160 nM PMA (Phorbol-12-myristate-13-acetate) (#524400, Sigma-Aldrich) for 24 hours, after which undifferentiated macrophages were removed. Foam cell formation was induced by incubating macrophages for 48 hours with oxLDL (80 \u0026micro;g/ml) (L34358, Thermo Fisher Scientific, MA, USA) or OxLDL plus VCE-005.1. After fixation, a Nile Red solution (N3013, Sigma-Aldrich) was used to stain the cells for 20 minutes. Samples were prepared with Vectashield Mounting Medium with DAPI, and a fluorescence microscope Leica Thunder Imager 3D assay (LEICA, L\u0026rsquo;Hospitalet de Llobregat, Spain) was used to acquire images.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eTHP-1 cells (5 \u0026times; 10⁵ cells/well) were plated in 24-well plates and treated with VCE-005.1 for 16 hours. Afterward, the cells were collected using Trypsin-EDTA (0.25%), centrifuged for 5 minutes (3500 rpm, 4\u0026deg;C), and permeabilized in 700 \u0026micro;L of 70% ethanol at 4\u0026deg;C overnight. Following washing, the cells were incubated with PBS containing 1 mg/mL propidium iodide and 5 \u0026micro;L/mL RNase for 3 hours under orbital shaking at room temperature in the dark. Finally, samples were examined via flow cytometry with a BD FACSCanto II flow cytometer (Beckton, Dickinson and Company, New Jersey, USA) to determine PE-A expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMice used in the study were sourced from Charles River Laboratories (Barcelona, Spain) and kept with unrestricted access to standard food and water in a controlled environment (temperature 20 \u0026ordm;C (\u0026plusmn;\u0026thinsp;2 \u0026ordm;C), 40\u0026ndash;50% relative humidity, and a 12-hour light/dark cycle). The experiments were conducted in accordance with European Union regulations and received approval from the Animal Research Ethics Committee of Cordoba University under Project Numbers 01/06/2020/072 and 31/03/2022/057.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMouse aortic ring assay\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice were treated daily intraperitoneally (i.p.) either with vehicle (ethanol: cremophor: saline solution 1:1:18) or with VCE-005.1 (30 mg/kg and 60 mg/kg). After seven days of treatment, the thoracic aorta was sectioned into 1 mm aortic rings and cultured in Opti-MEM (51985-034, Thermo Fisher Scientific) supplemented with 1% (v/v) penicillin/streptomycin in a humidified incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e and images of the samples were collected at day 10.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCritical limb ischemia (CLI) and vascular casting.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMale C57BL/6 mice (aged 10\u0026ndash;12 weeks) were assigned to two groups, and femoral ligations were performed as previously described [10]. VCE-005.1 (60 mg/kg) was administered i.p. daily throughout the duration of the study. The control group received the vehicle treatment. 10 days later, the mice were anaesthetized and administered 1000 \u0026micro;l of heparin (#9041-08-1, Sigma-Aldrich). They were then perfused and the limb muscles were excised. Following this, the samples were frozen using isopentane cooled in liquid nitrogen. Subsequently, the samples were cut into 5 \u0026micro;m sections at -21\u0026deg;C using a Leica CM1950 Cryostat (Leica Microsystems, Wetzlar, Germany). Vascular casting was performed using Microfil (MV-112 (white), Flow Tech Inc., South Windsor, CT, USA) as indicated in [10].\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ewound healing experiments.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMale 8-week-old diabetic db/db mice and their normoglycemic heterozygous db/+ controls were divided into 4 groups (4\u0026ndash;6 mice/group): db/+ + vehicle group; db/+ + VCE-005.1, db/db\u0026thinsp;+\u0026thinsp;vehicle group and db/db\u0026thinsp;+\u0026thinsp;VCE-005.1. On the day of injury, the mice were anesthetized with isoflurane, and their dorsal hair was removed using an electric razor and depilatory cream. The area was disinfected with alcohol, and two 6 mm wounds were created using a biopsy punch. A rubber splint made of silica gel (internal and external diameters of 8 and 15 mm, respectively) was placed around the wound to prevent closure of the native wound. The splint was secured using SuperGlue and fixed with 4\u0026ndash;6 interrupted sutures. 50 \u0026micro;L of VCE-005.1 treatment (cream containing 1% of compound, 5% liquid petrolatum, and 94% solid petrolatum) or vehicle (cream with 100% of solid petrolatum) were applied topically to the wound site every two days using a syringe and a transparent bandage (Tegadem 3M) was used to cover the wound. Digital photographs were taken every two to three days over a period of 10 days. The wound area was measured in pixels using a ruler and a rubber splint, and the analysis was performed using ImageJ 1.32 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsbweb.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"http://rsbweb.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The results were presented as the percentage of wound healing on the days evaluated. Finally, tissue samples were collected, with one wound reserved for histological analysis and the other for mRNA and protein expression studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin sections (5 \u0026micro;m) were processed and examined using H\u0026amp;E staining or incubated with Harris Hematoxylin, Acid Fuchsine, Phosphomolybdic acid, and 1% Acetic acid for Masson's Trichrome staining. Skin samples from each mouse were analysed using a semi-quantitative scoring method to assess re-epithelialisation, epithelial thickness index, remodelling and new collagen formation, as described previously [23]. For immunohistochemical (IHC) analysis, tissue sections (5 \u0026micro;m thick) were deparaffinised and subjected to boiling treatment in 1x citrate buffer for 10 minutes, followed by incubation in a solution of 3.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol for 10 minutes. Subsequently, the samples were washed three times with PBS containing 0.1% Tween, treated with a blocking solution (#20773, Merck, Darmstadt, Germany) and left to incubate at 4\u0026deg;C overnight with the corresponding primary antibody (Supplemental Table\u0026nbsp;1). After that the slides were incubated 1h RT with the appropriate secondary antibody (Supplemental Table\u0026nbsp;1). Following this, the samples were treated with avidin-biotin-peroxidase reagent (PK-6100, Vector Laboratories) and DAB (3,3'-diaminobenzidine) chromogen (K346811-2, Dako, Santa Clara, CA, USA). The preparations were then counterstained with hematoxylin followed by dehydration and mounted with Eukitt mounting medium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConfocal immunofluorescence.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGastrocnemius muscle samples (5 \u0026micro;m thick) were fixed for 10 minutes at -20\u0026deg;C in a 1:1 methanol-acetone solution, while skin wound samples (5 \u0026micro;m thick) were deparaffinized and prepared for immunofluorescence as described [10]. The samples were then left to incubate overnight at 4\u0026deg;C with the appropriate primary antibody (Supplemental Table\u0026nbsp;1). The following day, after performing three washes, the secondary antibody was incorporated (Supplemental Table\u0026nbsp;1). After mounting the slides with Vectashield Mounting Medium with DAPI, images were obtained using a LSM710 laser scanning confocal spectral microscope (Zeiss) with either a 20x/0.8 or 40x oil immersion Plan-Apochromat objective.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and qRT-PCR\u003c/h2\u003e \u003cp\u003eSkin wound tissue and gastrocnemius muscle samples were collected for RNA extraction using the RNeasy Lipid Mini Kit (#74804, Qiagen, Hilden, Germany), in accordance with the manufacturer's instructions. A total of 1 \u0026micro;g of RNA was then reverse transcribed into complementary DNA (cDNA) via the iScript cDNA Synthesis Kit (#1708891, Bio-Rad, Hercules, CA, USA). The resulting cDNA was then subjected to quantitative real-time PCR (qPCR) using the iQ\u0026trade; SYBR Green Supermix (#1708880, Bio-Rad, Hercules, CA, USA) and a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). PCR efficiency was normalized to the expression of the PPIA gene for each sample, and the relative fold change in gene expression was determined using the -ΔΔCt approach. The primers employed for amplification were synthesized by Eurofins (Luxembourg) and are detailed in the Supplemental Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOlink Proximity Extension Assay (PEA).\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePlasma samples from different animal groups were submitted to Cobiomic Biosciences SL (C\u0026oacute;rdoba, Spain), for detection of proteins across the Olink Target 96 Mouse panel (Uppsala, Sweden). Data from Olink PEA were presented as normalized protein expression (NPX) values on a Log2 scale, and their intensity was adjusted using the plate median for each assay to minimize both intra- and inter-assay variation. NPX data was used to detect variations in individual protein levels across samples and helped establish protein signatures by relatively quantifying protein levels. For a list of proteins included in the mouse exploratory panel please visit: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cobiomicbioscience.com/wp-content/uploads/2024/04/Target-96-Mouse-Exploratory.pdf\u003c/span\u003e\u003cspan address=\"https://cobiomicbioscience.com/wp-content/uploads/2024/04/Target-96-Mouse-Exploratory.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll the images were analyzed using the ImageJ software and data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM or SD. The Kolmogorov-Smirnov, Shapiro-Wilk or D'Agostino tests and Pearson's test were applied to evaluate the normality of the data. One-way analysis of variance (ANOVA) was then performed, followed by Tukey's or Dunnett's post-hoc tests for parametric comparisons, or the Kruskal-Wallis test for non-parametric analyses. A significance level of p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered. All statistical analyses were conducted using GraphPad Prism version 9 (GraphPad, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eVCE-005.1 stabilizes HIF-1α through a pathway that involves SIRT1 and AMPK and prevents endothelial senescence\u003c/h2\u003e \u003cp\u003eIn previous studies, we demonstrated that the compound VCE-005.1 activates HIF-1α through a PP2A/B55α-dependent pathway, with AMPK signalling also playing a role in is the mechanism of action [17, 21]. AMPK is known for its cardiovascular protective effects, regulating endothelial function, redox homeostasis, and inflammation [23]. Herein we have further examined the effect of VCE-005.1 on AMPK activation and downstream targets such as SIRT1 and eNOS. We found that VCE-005.1 induced AMPK phosphorylation, SIRT1 expression and HIF-1α stabilization in vascular endothelial cells. Dosomorphin (DS), an AMPK inhibitor, blunted VCE-005.1-induced AMPK phosphorylation and SIRT1 expression and attenuated HIF-1α stabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Furthermore, knocking down SIRT1 expression (siSIRT1) greatly prevented VCE-005.1-induced HIF-1α activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe interaction between AMPK and SIRT1 is bidirectional, as AMPK can activate SIRT1 by increasing NAD\u003csup\u003e+\u003c/sup\u003e levels in cells, thereby promoting its activation. In turn, SIRT1 deacetylates the AMPK upstream kinase LKB1, leading to AMPK phosphorylation and activation [24\u0026ndash;26]. We observed that treatment with VCE-005.1 increased both SIRT1 enzymatic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) and the NAD\u003csup\u003e+\u003c/sup\u003e/NADH ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) in EA.hy926 cells. Next, we evaluated the effect of VCE-005.1 on cellular senescence in HMEC-1 cells exposed to oxidative stress induced by H₂O₂. Treatment with VCE-005.1 reduced SA-β-gal\u003csup\u003e+\u003c/sup\u003e expression, a hallmark of senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). In addition, VCE-005.1 treatment restored SIRT1 levels, which were diminished by H₂O₂, while downregulating p21, a key senescence marker, which was upregulated in response to oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). These findings suggest that VCE-005.1 effectively counteracts endothelial senescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain further insight into the mechanism of action of VCE-005.1 on the AMPK/SIRT1/HIF-1α axis, a transactivation luciferase assay was performed in NIH-3T3-EPO-Luc cells treated with VCE-005.1 either in the absence or in the presence of specific inhibitors of AMPK (DS), SIRT1 (EX527), and HSP90 (CCT018159), the latter being a chaperone that interacts with AMPK [27] and associates with HIF-1α to facilitate its nuclear accumulation [28]. All these inhibitors significantly prevented VCE-005-1-induced EPO-Luc transactivation as a surrogated marker of HIF activation. Additionally, we observed that CCT018159 also inhibited HIF-1α and SIRT1 induction in VCE-005-1-treated endothelial cells (Supplemental Fig.\u0026nbsp;1a and 1b).\u003c/p\u003e \u003cp\u003eThe impact of VCE-005.1 on eNOS phosphorylation at Ser1177, another key target of AMPK [29] and its relation with B55α was also investigated. Our results demonstrated that VCE-005.1 promoted eNOS phosphorylation, which was significantly reduced in cells knocked down for B55α by siRNA (Supplemental Fig.\u0026nbsp;1c). These findings highlight the functional interaction between B55α and AMPK in response to VCE-005.1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVCE-005.1 inhibits the formation of foam cells and induces apoptosis in macrophages.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOxidized low-density lipoproteins (ox-LDL) induce an overaccumulation of lipids in macrophages, resulting in the formation of foam cells, a key event in the onset and advancement of atherosclerosis. These cells contribute significantly to the development of atherosclerotic plaques, serving as a major source of the necrotic core [30, 31]. We investigated the effect of VCE-005.1 on ox-LDL-induced foam cell formation and found that it significantly reduced lipid accumulation in ox-LDL-treated macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eMild hypoxia induces macrophage apoptosis, contributing to the regulation of inflammation in diseases such as multiple sclerosis (MS) [32]. Consequently, small hypoxia-mimetic compounds may have similar effects on macrophages. Given their key role in the pathophysiology of atherosclerosis, we investigated whether VCE-005.1 induces macrophage apoptosis. Our results show that VCE-005.1 promotes PARP-1 fragmentation, which coincides with HIF-1α induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Furthermore, cell cycle analysis revealed a significant increase in the percentage of sub-diploid cells following VCE-005.1 treatment, indicating enhanced apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of VCE-005.1 on vasculogenesis\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the influence of VCE-005.1 on vascularization and sprouting, an \u003cem\u003eex vivo\u003c/em\u003e aortic ring assay was performed. The results showed that VCE-005.1 treatment significantly increased the formation of new sprouts, suggesting its ability to promote vascularization \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To further investigate its effects \u003cem\u003ein vivo\u003c/em\u003e, a critical limb ischemia (CLI) model was established by performing a double ligation of the left femoral artery. The results showed that VCE-005.1 treatment clearly enhanced arterialization in the limb with the clamped artery without affecting the vascular structure of the control limb (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Additionally, the expression mRNA levels of HIF-1α target genes essential for angiogenesis, including hepatocyte growth factor (\u003cem\u003eHgf\u003c/em\u003e), erythropoietin (\u003cem\u003eEpo\u003c/em\u003e), and vascular endothelial growth factor A (\u003cem\u003eVegf-A\u003c/em\u003e), were also analysed in the gastrocnemius muscle. The results indicated that the expression levels of these genes were significantly higher in the ligated limb treated with VCE-005.1 compared to the untreated ligated limb. However, this significant induction was not observed in the healthy limb treated with the compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCaveolin-1 (CAV1), regulated by HIF-1α, plays a essential role in angiogenesis and the vascular response to ischemia [33, 34]. In our study, CAV1 expression was reduced in the ligated limb of untreated CLI mice. However, treatment with VCE-005.1 restored its expression levels, suggesting a protective role in the affected limb (Supplemental Fig.\u0026nbsp;2a). Moreover, VCE-005.1 stimulated the formation of mature vessels (CD31\u003csup\u003e+\u003c/sup\u003e/αSMA\u003csup\u003e+\u003c/sup\u003e) and promoted endothelial cell proliferation (CD31\u003csup\u003e+\u003c/sup\u003e/Ki67\u003csup\u003e+\u003c/sup\u003e), specifically in the vascular endothelium of the ischemic limb (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). These findings indicate that VCE-005.1 acts selectively in hypoxic tissues. Moreover, we examined the expression of SIRT1 in the gastrocnemius of CLI mice and we found a decrease in SIRT1 expression in the clamped limb that was reversed in mice treated with VCE-005.1, without affecting the control limb (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the effects of VCE-005.1 on peripheral biomarkers associated with CLI, the compound was administered i.p. to CLI mice for 7 days and plasmatic biomarkers were analysed using a proximity extension assay. In CLI mice, we observed significant alterations in inflammatory, endothelial, apoptotic, oxidative stress-related, and vascular remodelling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The inflammatory response was characterized by increased levels of tumour necrosis factor-alpha (TNF-α), C-C motif chemokine ligand 2 (CCL2/MCP-1), C-C motif chemokine ligand 3 (CCL3/MIP-1α), and interleukin-10 (IL-10), indicating an imbalance between pro-inflammatory and compensatory anti-inflammatory mechanisms.\u003c/p\u003e \u003cp\u003eEndothelial dysfunction and vascular remodelling were evidenced by the upregulation of platelet-derived growth factor subunit B (PDGFB) and WNT1-inducible signalling pathway protein 1 (WISP1), suggesting excessive vascular remodelling and fibrosis. Apoptotic and senescence-associated changes were reflected in the increased expression of FAS cell surface death receptor (FAS), poly [ADP-ribose] polymerase 1 (PARP1), and tumour necrosis factor receptor superfamily member 11B (TNFRSF11B/osteoprotegerin, OPG), suggesting heightened cell death and vascular aging. The oxidative stress response was also altered, as indicated by increased levels of peroxiredoxin-5 (PRDX5), which may represent an attempt to counteract oxidative damage.\u003c/p\u003e \u003cp\u003eNeurovascular dysfunction and impaired regenerative capacity were reflected in the upregulation of follistatin (FST) and hepatocyte growth factor (HGF), likely as a response to promote muscle repair and angiogenesis.\u003c/p\u003e \u003cp\u003eIn VCE-005.1-treated CLI mice, we observed a restoration of inflammatory balance, as levels of TNF-α, CCL2, CCL3, and IL-10 returned to control values. Endothelial stability was improved through modulation of PDGFB and WISP1, suggesting a more controlled vascular remodelling process. Apoptotic and senescence-associated markers FAS, PARP1, and TNFRSF11B were normalized, indicating a protective effect against cell death and vascular aging. The oxidative stress response remained stable with PRDX5 levels maintained, reflecting a sustained antioxidant defence. Neurovascular recovery was enhanced with the further increase of FST and HGF, reinforcing the potential of VCE-005.1 to promote vascular repair and tissue regeneration.\u003c/p\u003e \u003cp\u003eThese findings suggest that VCE-005.1 mitigates CLI-induced inflammation, apoptosis, and vascular senescence while enhancing endothelial function and neurovascular regeneration, highlighting its potential as a therapeutic strategy in peripheral ischemic conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVCE-005.1 protects endothelial cells against glucose-induced cytotoxicity and enhances wound healing in diabetic mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePAD and DFUs arise from hyperglycemia-induced endothelial dysfunction, promoting atherosclerosis, reduced blood flow, and impaired healing, increasing the risk of complications [35, 36]. To investigate the effect of VCE-005.1 on cell viability under high glucose conditions, EA.hy926 cells were exposed to 270 mM glucose with or without the compound. VCE-005.1 significantly improved cell viability under these conditions, indicating a protective effect against glucose-induced cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTo assess the efficacy of VCE-005.1 in diabetic wound healing \u003cem\u003ein vivo\u003c/em\u003e, we examined its effects in diabetic db/db mice and their db/+ controls. VCE-005.1 (1% in Vaseline) was applied locally every other day for 10 days, significantly accelerating wound closure compared to the vehicle-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). This improvement was observed in both heterozygous and homozygous diabetic mice, although untreated diabetic mice exhibited slower wound healing. We also found that topical VCE-005.1 promoted re-epithelialization and increased the skin thickness index 10 days post-injury in both experimental groups. Additionally, tissue remodelling was enhanced, and collagen deposition in the wound area was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePersistent inflammation is a hallmark of impaired wound healing in diabetes and plays a critical role in tissue repair dysfunction. To assess the impact of VCE-005.1 on immune cell recruitment at the wound site, macrophage and neutrophil infiltration were analysed. VCE-005.1-treated mice exhibited a significant reduction in macrophages, as indicated by F4/80 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec), and neutrophils, as detected by MPO staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), compared to vehicle-treated mice. This effect was observed in both homozygous and heterozygous diabetic animals, suggesting a modulation of the inflammatory response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the pro-angiogenic effects of VCE-005.1 in wound healing, vessel density and perimeter were quantified using CD31\u003csup\u003e+\u003c/sup\u003e/αSMA\u003csup\u003e+\u003c/sup\u003e staining, alongside the expression of the endothelial markers CD31 and CD34. VCE-005.1 treatment significantly increased microvessel density and perimeter in the wound area (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Moreover, the number of double-stained vessels (CD34\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e) was elevated, indicating the induction of de novo vessel formation. These angiogenic effects were evident in both control and diabetic mice treated with VCE-005.1.\u003c/p\u003e \u003cp\u003eThe expression mRNA levels of HIF-1α target genes essential for angiogenesis (\u003cem\u003eHGF, Epo, Vegf-a\u003c/em\u003e) were also analysed in wounded skin tissue. Their expression was significantly reduced in untreated groups compared to VCE-005.1-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb), suggesting a role for VCE-005.1 in promoting HIF-1α-mediated angiogenesis. In addition, CAV1 expression, another gene regulated by HIF-1α, was significantly increased following VCE-005.1 treatment in both db/db and db/+ mice. Notably, CAV1 expression was specifically localized to endothelial cells, as confirmed by double staining for CAV1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e. Furthermore, VCE-005.1 treatment not only increased CAV1 protein expression but also significantly upregulated its mRNA levels (Supplemental Fig.\u0026nbsp;2b, 2c and 2d), reinforcing its role in endothelial function and vascular remodelling.\u003c/p\u003e \u003cp\u003eThese findings suggest that VCE-005.1 enhances wound healing by reducing excessive inflammation and promoting angiogenesis through HIF-1α signalling in endothelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVCE-005.1 induces the expression of B55α and HIF-1α in endothelial cells\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs previously demonstrated, our data establish a mechanistic link between B55α and SIRT1 expression and activity \u003cem\u003ein vitro\u003c/em\u003e. To further investigate this relationship, we analysed their expression in skin samples following injury. Topical application of VCE-005.1 significantly upregulated B55α and SIRT1 expression in the dermal wound area of both diabetic and control mice compared to untreated controls, suggesting a role in wound healing. Notably, B55α and SIRT1 expression was specifically localized to endothelial cells, as confirmed by double staining for B55α\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e and SIRT1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). Furthermore, mRNA levels of both proteins were significantly elevated in the dermis of VCE-005.1-treated db/+ and db/db mice post-injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). These findings suggest that VCE-005.1 promotes endothelial-specific B55α and SIRT1 expression, potentially contributing to its pro-reparative and angiogenic effects during wound healing.\u003c/p\u003e \u003cp\u003eAdditionally, we found that VCE-005.1 enhanced hair follicle generation, particularly in control mice, suggesting a role in stimulating hair follicle-associated regenerative processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Hair follicles play a fundamental role in skin tissue repair and wound healing by serving as a reservoir of stem cells and secreting growth factors essential for tissue regeneration. Interestingly, HIF-1α plays a key role in hair follicle regeneration and dermal vascularization [9, 37]. Given that hair dermal papilla cells (HDPCs) are critical for hair follicle regeneration and may contribute to wound healing, we evaluated whether VCE-005.1 could stimulate HIF-1α expression in immortalized HDPC cells (IHDPCs). Our results demonstrated that VCE-005.1 activates both B55α and HIF-1α in a dose-dependent manner, as confirmed by Western blot (Supplemental Fig.\u0026nbsp;3a) and immunofluorescence (Supplemental Fig.\u0026nbsp;3b).\u003c/p\u003e \u003cp\u003eThese findings suggest that VCE-005.1 enhances B55α and HIF-1α expression in endothelial cells and dermal papilla cells, reinforcing its potential to promote vascularization, hair follicle regeneration, and overall skin tissue repair.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePAD and DFUs result from impaired angiogenesis and endothelial dysfunction, which restrict blood flow and compromise tissue healing. Effective revascularization is therefore crucial for restoring circulation and promoting repair. While gene therapies targeting angiogenic factors like VEGF and stem cell-based approaches have shown promise, their clinical application remains limited due to efficacy challenges, safety concerns, and difficulties in achieving sustained effects [38, 39]. These limitations highlight the need for alternative or complementary strategies to enhance vascular regeneration and improve patient outcomes.\u003c/p\u003e \u003cp\u003eIn this context, our study investigates the potential of VCE-005.1 as a therapeutic agent for endothelial protection and vascular regeneration. Our findings provide evidence that VCE-005.1 exerts significant protective and regenerative effects in preclinical models of critical limb ischemia (CLI) and diabetic wound healing, likely mediated by the activation of the B55α/AMPK/SIRT1/HIF-1α axis, which represent key pathway(s) involved in endothelial function, angiogenesis, prevention of endothelial senescence, and tissue repair.\u003c/p\u003e \u003cp\u003eThe B55α regulatory subunit of PP2A plays a dual role in mitotic regulation and hypoxia signaling. In mitosis, it ensures cell cycle progression by dephosphorylating key substrates involved in chromosome segregation, nuclear envelope reformation, and anaphase-promoting complex regulation [40, 41]. Simultaneously, B55α interacts with PHD2, modulating its activity through dephosphorylation at Ser125, thereby influencing HIF-1α stability and cellular adaptation to hypoxia [12]. Our results suggest that B55α/PHD2 interaction is one of the primary targets for the vasculogenic activity of VCE-005.1, as no mitotic effects were observed in vitro, despite enhanced vascular endothelial proliferation in vivo. This indicates that vascular regeneration is primarily driven by hypoxia-induced signaling rather than direct mitotic stimulation, with the HIF-1α/VEGF axis likely mediating endothelial expansion and vascular remodeling.\u003c/p\u003e \u003cp\u003eProteomic analysis of the B55α interactome has identified numerous substrates, predominantly proteins involved in mitotic regulation [42]. Notably, AMPK, SIRT1, and PHD2 were not detected in these analyses, despite prior evidence supporting a functional interaction between B55α and PHD2 [12]. This discrepancy may stem from technical limitations of proteomic methods, which often favor the detection of stable or abundant interactions and may overlook transient, low-abundance, or context-specific complexes. Therefore, the absence of these proteins in the interactome should not be interpreted as evidence of a lack of interaction, but rather as a limitation of the methodology. This reinforces the need for complementary approaches to fully elucidate the regulatory network of B55α.\u003c/p\u003e \u003cp\u003eIn response to B55α activators, such as VCE-005.1 and Etrinabdione, siRNA-mediated B55α knockdown prevents HIF-1α activation, mirroring the effects observed with siRNA targeting SIRT1 and pharmacological inhibition of AMPK and SIRT1 [17, 43]. Notably, other AMPK activators, such as metformin, and SIRT1 activators, such as resveratrol, fail to induce HIF-1α expression. While SIRT1 has been reported to repress HIF-1α activity through deacetylation and inhibition of coactivator recruitment [44], our findings suggest that in the context of VCE-005.1 treatment, SIRT1 may contribute to HIF-1α stabilization in cooperation with AMPK and HSP90. These dual roles highlight the context-dependent nature of the influence of SIRT1 on HIF-1α, suggesting that its effect may vary according to cellular environment, metabolic status, or cofactor availability. Our findings suggest that B55α may function as a central regulatory node in two distinct but interconnected pathways: in one, B55α/PHD2 initiates HIF-1α protein stabilization, while in the other, B55α/AMPK/SIRT1 enhances such stabilization and induces HIF-1α nuclear translocation in coordination with HSP90 [28]. These findings position B55α as a critical modulator of HIF-1α signaling, integrating B55α-mediated inhibition of PHD2 and B55α/AMPK/SIRT1 signalling with significant implications for HIF-1α activation, hypoxia adaptation and vascular remodeling.\u003c/p\u003e \u003cp\u003eThe precise nature of the interactions between B55α, AMPK, and SIRT1 remains unclear. Direct interaction appears unlikely, as PP2A-mediated AMPK activation by dephosphorylation has not been described. On the contrary, PP2A is generally known to inactivate AMPK by dephosphorylating Thr172, a key activating site [45]. However, the regulation of AMPK is complex and involves multiple phosphorylation sites. Inhibitory phosphorylation can occur at serine residues such as Ser485 on the α1-subunit (or Ser491 on the α2-subunit), which can prevent activation by hindering phosphorylation at Thr172. It is therefore conceivable, though speculative, that under specific conditions, PP2A/B55α might dephosphorylate these inhibitory serine residues, thereby facilitating subsequent activation at Thr172 by upstream kinases such as LKB1, CaMKKβ, or TAK1[46]. Further experimental validation is needed to support this hypothesis.\u003c/p\u003e \u003cp\u003eSimilarly, no direct regulation of SIRT1 by PP2A has been demonstrated, although indirect regulatory mechanisms may exist. Indeed, proteomic analysis of the B55α interactome has identified numerous substrates, predominantly proteins involved in mitotic regulation [42, 47]. However, AMPK, SIRT1, and PHD2 were not detected, despite prior evidence supporting a direct interaction between B55α and PHD2 [12]. This discrepancy may be due to technical limitations of interactome analysis, which may favor the detection of specific protein subsets, potentially overlooking transient or context-dependent interactions. Further investigation is warranted to determine whether inhibition of B55α interaction with selected substrates as described by Hein et al. [42] can suppress HIF activation and angiogenesis induced by VCE-005.1 or other B55α activators, potentially unveiling new regulatory mechanisms of B55α in hypoxia-driven vascular remodeling. Additionally, we cannot rule out the possibility that VCE-005.1 influences the phosphorylation status of endogenous inhibitors of PP2A/B55α, such as Arspp19, which is regulated by the Greatwall kinase [48].\u003c/p\u003e \u003cp\u003eVCE-005.1 also demonstrated the ability to inhibit foam cell formation and induce apoptosis in macrophages, key contributors to atherosclerotic plaque progression [49]. By reducing lipid accumulation in ox-LDL-treated macrophages, VCE-005.1 may attenuate the formation of necrotic cores within atherosclerotic plaques. Its capacity to induce apoptosis in macrophages, likely through the HIF-1α pathway [50], suggests a potential therapeutic avenue for controlling inflammatory responses in vascular diseases. Additionally, its effect on PARP-1 cleavage supports its role in promoting programmed cell death, reinforcing its anti-atherosclerotic properties.\u003c/p\u003e \u003cp\u003eThe compound significantly enhanced vascularization and arteriogenesis in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models. The \u003cem\u003eex vivo\u003c/em\u003e aortic ring assay revealed increased new sprout formation, while the CLI model confirmed improved collateral vessel formation following VCE-005.1 treatment. The upregulation of angiogenesis-related genes, including \u003cem\u003eHgf\u003c/em\u003e, \u003cem\u003eEpo\u003c/em\u003e, and \u003cem\u003eVegf-A\u003c/em\u003e, further supports the pro-angiogenic activity of VCE-005.1. Notably, its effects were selective for ischemic tissues, as no significant changes were observed in non-ligated limbs, highlighting the ability of the treatment to act in hypoxic tissues. It also promoted the formation of mature blood vessels and enhanced endothelial cell proliferation, demonstrating its efficacy in promoting functional neovascularization. This effect correlated with increased expression of Caveolin-1, a HIF-1α-dependent gene that may play a key role in endothelial function.\u003c/p\u003e \u003cp\u003eChronic hyperglycemia is a major contributor to endothelial dysfunction and impaired wound healing in diabetic patients. Our results show that VCE-005.1 significantly improved endothelial cell viability under high glucose conditions, suggesting a protective effect against glucose-induced cytotoxicity. In a diabetic wound healing model, the compound significantly accelerated wound closure, promoted re-epithelialization, and enhanced tissue remodelling. The observed increase in collagen deposition and microvessel density suggests that VCE-005.1 fosters a pro-healing environment. Additionally, it reduced macrophage and neutrophil infiltration at the wound site, indicating its anti-inflammatory properties. These findings reinforce its therapeutic potential for improving vascular and tissue repair processes in diabetic patients.\u003c/p\u003e \u003cp\u003eOur study also highlights the crucial role of B55α and HIF-1α in the mechanism of action of VCE-005.1. Increased expression of B55α and SIRT1 in endothelial cells was observed both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e following treatment. The selective activation of these proteins in the skin of diabetic mice suggests that VCE-005.1 enhances endothelial-specific responses, promoting tissue regeneration and vascular repair. For instance, the induction of HIF-1α in hair dermal papilla cells suggests a potential role in skin regeneration, which may have broader implications for wound healing and dermatological applications [51, 52].\u003c/p\u003e \u003cp\u003eIn summary, our findings establish VCE-005.1 as a promising therapeutic candidate for peripheral artery disease, critical limb ischemia, and diabetic foot ulcers by targeting the B55α/AMPK/SIRT1/HIF-1α axis. The compound promotes endothelial protection, inhibits foam cell formation, enhances angiogenesis and arteriogenesis, protects against hyperglycemia-induced cytotoxicity, and accelerates wound healing. Its selective action in ischemic and damaged tissues underscores its therapeutic potential for vascular and metabolic diseases. Future studies should focus on further characterizing its pharmacokinetic properties and clinical translation to optimize its therapeutic application in ischemic vascular diseases.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, VCE-005.1 exhibits potent regenerative and cytoprotective effects in preclinical models of peripheral artery disease, critical limb ischemia, and diabetic foot ulcers. The compound enhances angiogenesis, promotes endothelial proliferation, reduces inflammation, and protects against hyperglycemia-induced cytotoxicity via activation of the B55α/AMPK/SIRT1/HIF-1α axis. Notably, VCE-005.1 selectively targets hypoxic and ischemic tissues, minimizing effects in normoxic regions. Systemic biomarker profiling further supports its therapeutic action, showing restoration of inflammatory balance, attenuation of endothelial dysfunction, normalization of apoptotic and senescence markers, and enhancement of neurovascular recovery. These findings underscore its ability to modulate key pathogenic mechanisms, including vascular remodeling, oxidative stress, and immune dysregulation, associated with CLI. By targeting both cellular and systemic pathways involved in vascular damage and repair, VCE-005.1 emerges as a promising candidate for the treatment of ischemic and metabolic vascular diseases. Further studies are warranted to optimize its clinical translation and explore its long-term efficacy and safety.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"784\" style=\"margin-right: calc(12%); width: 88%;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eAMPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 20.3642%;\"\u003e\n \u003cp\u003eAMP-activated protein kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eB55\u0026alpha; PP2A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 680px;\"\u003e\n \u003cp\u003eProtein phosphatase 2A-B55\u0026alpha; subunit (PPP2R2A)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eBAH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 680px;\"\u003e\n \u003cp\u003eBetulinic acid hydroxamate\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eBSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003eBovine serum albumin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eCAV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003eCaveolin 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eCCL2/MCP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003eC-C motif chemokine ligand 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eCCL3/MIP-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003eC-C motif chemokine ligand 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ecDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eComplementary DNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eCLI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eCritical limb ischemia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eDFU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eDiabetic foot ulcers\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eDorsomorphin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eECs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eEndothelial cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eEGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eEpidermal growth factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eeNOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eEndothelial nitric oxide synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eEPO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eErythropoietin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eFAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eFAS cell surface death receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eFBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eFetal bovine serum\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eFST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eFollistatin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eH\u0026amp;E\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003eHematoxylin and Eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eHDPCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003eHair dermal papilla cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eHG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eHigh glucose\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eHgf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eHepatocyte growth factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eHIF-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eHypoxia-inducible factor 1-alpha\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eHMEC-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eHuman microvascular endothelial cells\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ei.p.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eintraperitoneally\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eIF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eImmunofluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eIHC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eImmunohistochemical\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eIHHDPC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eImmortalized Human Hair Dermal Papilla Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eMTT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003e3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eNicotinamide adenine dinucleotide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eNADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eNicotinamide adenine dinucleotide hydrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eNPX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003eNormalized Protein Expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eOx-LDL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eOxidized low-density lipoprotein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ePAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003ePeripheral artery disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ePARP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003ePoly(ADP-ribose) polymerase-1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003ePhosphate buffered saline\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ePEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eProximity extension assay\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ePRDX5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003ePeroxiredoxin-5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ePHD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eProlyl hydroxylase 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ePHDs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eProlyl hydroxylases\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003ePMA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003ePhorbol-12-myristate-13-acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eQuantitative real-time PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eReactive oxygen species\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eStandard deviation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eSIRT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eSirtuin 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eTBI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70.827%;\"\u003e\n \u003cp\u003eTraumatic brain injury\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eTBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eTris-buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eTNFRSF11B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eOsteoprotegerin, OPG. Tumour necrosis factor receptor superfamily member 11B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eTNF\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eTumour necrosis factor-alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eVCE-004.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eEtrinabdione or EHP-101, [(1\u0026prime;R,6\u0026prime;R)-3-(benzylamine)-\u003c/p\u003e\n \u003cp\u003e6-hydroxy-3\u0026prime; -methyl -4-pentyl -6\u0026prime; - (prop-1-en-2-yl )\u003c/p\u003e\n \u003cp\u003e[1,1\u0026prime;bi(cyclohexane)]-2\u0026prime;,3,6-triene-2,5-dione)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eVCE-005.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eBAH or 3\u0026beta;-hydroxylup-20(29)-en-28-oic acid hydroxamate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eVEGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003evascular endothelial growth factor\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eVegfa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eVascular endothelial growth factor A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.2813%;\"\u003e\n \u003cp\u003eWISP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85.0971%;\"\u003e\n \u003cp\u003eWNT1-inducible signalling pathway protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Declarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols followed the guidelines of animal care set by the EU guidelines 86/609/EEC, the Ethic Committee on Animal Experimentation at the University of C\u0026oacute;rdoba (Spain) and the Andalusian Regional Committee for Animal Experimentation approved all the procedures described in this study (31/03/2022/057).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grant CPP2021-008557/AEI/10.13039/501100011033/ Uni\u0026oacute;n Europea NextGenerationEU/PRTR, grant PID2023-148340OB-I00 (Agencia Estatal de Investigaci\u0026oacute;n, Spain; co-funded with EU funds from FEDER Program), and CNS2022-135922/ Uni\u0026oacute;n Europea NextGenerationEU/Plan de Recuperaci\u0026oacute;n, Tranformaci\u0026oacute;n y Resilencia, Agencia Estatal de Investigaci\u0026oacute;n.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eILC, MEP, JJFS, IM, and AGM performed the\u003cem\u003e\u0026nbsp;\u003c/em\u003eexperiments and data analysis. AGM and EM designed the overall study, managed and supervised the study. ILC and EM wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the Advanced Optical Microscopy Unit and the Animal Experimentation Facilities of the IMIBIC.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBonaca MP, Hamburg NM, Creager MA (2021) Contemporary Medical Management of Peripheral Artery Disease. Circ Res 128:1868\u0026ndash;1884. https://doi.org/10.1161/CIRCRESAHA.121.318258\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnnex BH, Cooke JP (2021) New Directions in Therapeutic Angiogenesis and Arteriogenesis in Peripheral Arterial Disease. 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Stem Cells Transl Med 3:1209\u0026ndash;1219. https://doi.org/10.5966/sctm.2013-0217\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"B55α activators, AMPK, Sirtuin 1, angiogenesis, diabetic foot","lastPublishedDoi":"10.21203/rs.3.rs-6319136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6319136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePeripheral artery disease (PAD) and diabetic foot ulcers (DFUs) are chronic ischemic conditions characterized by endothelial dysfunction, impaired angiogenesis, and tissue hypoxia. The regulatory phosphatase subunit B55α (PP2A) modulates PHD2/HIF-1α axis, supporting vascular homeostasis and repair. Here, we investigated the mechanism and therapeutic potential of VCE-005.1, a selective B55α activator and PHD2 inhibitor, in PAD and DFU preclinical models.\u003c/p\u003e \u003cp\u003eIn human endothelial cells, VCE-005.1 activated B55α/AMPK/SIRT1/HIF-1α axis by inducing AMPK phosphorylation, elevating intracellular NAD⁺ levels, upregulating SIRT1 expression and enzymatic activity, stabilizing HIF-1α, and enhancing eNOS phosphorylation. VCE-005.1 also prevented oxidative stress\u0026ndash;induced endothelial senescence by reducing p21 and restoring SIRT1 levels. In macrophages, it inhibited foam cell formation and induced apoptosis via PARP-1 fragmentation.\u003c/p\u003e \u003cp\u003eIn a murine critical limb ischemia (CLI) model, VCE-005.1 enhanced arteriogenesis, endothelial proliferation, and mature vessel formation in hypoxic muscle, while selectively upregulating angiogenic genes (\u003cem\u003eVegf-A, Hgf, Epo\u003c/em\u003e) and caveolin-1. Plasma biomarker analysis revealed that VCE-005.1 normalized markers of inflammation, endothelial dysfunction, apoptosis, vascular aging, and promoted neurovascular repair. In diabetic db/db mice, topical VCE-005.1 improved wound closure, re-epithelialization, collagen deposition, and microvascular density, while reducing neutrophil and macrophage infiltration. These effects correlated with localized induction of B55α and SIRT1 expression in endothelial and dermal papilla cells.\u003c/p\u003e \u003cp\u003eThese findings position VCE-005.1 as a promising tissue-selective therapeutic candidate for ischemic vascular diseases. By enhancing angiogenesis, preventing endothelial senescence, reducing cellular damage, and selectively targeting hypoxic tissues, VCE-005.1 may overcome the limitations of current pro-angiogenic therapies, offering new hope for patients with PAD and DFUs.\u003c/p\u003e","manuscriptTitle":"B55α Orchestrates AMPK/SIRT1/HIF-1α Signaling: VCE-005.1 as a Tissue-Selective Therapeutic Strategy for Ischemic Vascular Diseases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 11:42:20","doi":"10.21203/rs.3.rs-6319136/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b2f8c346-30d8-427b-a502-9f17b5e660dc","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-06T08:08:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-31 11:42:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6319136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6319136","identity":"rs-6319136","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}