Small Chemical Molecule CPP Promotes Angiogenesis in Surgically Created Severe Lower Limb Ischemia and Diabetes-Induced Limb Vascular Reduction Models

Abstract

Peripheral artery disease (PAD) often progresses to chronic limb-threatening ischemia (CLTI), leading to severe limb dysfunction and amputation. Angiogenic therapies using small molecules offer advantages over cell-based approaches. Here, we investigated the in vivo angiogenic effects of CPP, a small chemical molecule previously shown to induce differentiation of human dermal fibroblasts into vascular endothelial cells via the PHD2/HIF1α/HEY1 pathway. In a mouse model of critical limb ischemia (CLI), subcutaneous multipoint injections of CPP (1 or 10 mg/kg/day) for 14 days significantly enhanced blood perfusion in ischemic limbs by day 7, with the 1 mg/kg dose increasing capillary density in skin and muscle by day 14. High-dose CPP showed no organ toxicity. In diabetic db/db mice, intraperitoneal CPP (1 or 5 mg/kg/day) for 30 days restored capillary density in skin and gastrocnemius muscle, counteracting diabetes-induced vascular rarefaction. These findings demonstrate that CPP promotes in situ angiogenesis and perfusion recovery in ischemic and diabetic conditions, highlighting its potential as a therapeutic agent for PAD.

1. Introduction

Patients with peripheral artery disease (PAD) commonly experience chronic limb-threatening ischemia (CLTI) in the end stage, leading to severe functional impairment of the limbs, amputation, and even death [1]. Among the various disease factors contributing to PAD, diabetes is significantly associated with PAD risk, leading to more severe symptoms and a poorer prognosis. Promoting angiogenesis at ischemic sites and improving blood flow are key to the recovery of limbs from ischemic injury. Angiogenic therapies based on cells and small-molecule drugs are considered important and promising strategies [2]. Cell-based therapies can be classified based on cell source into allogeneic therapies and autologous therapies. Autologous cells offer the advantage of not requiring immunosuppression but may be limited by factors such as cell unavailability and scarcity due to autologous factors. Allogeneic cells, while offering a more abundant cell source, present drawbacks, including immune incompatibility and ethical concerns, which restrict their application. Small chemical molecules offer advantages such as convenience, low cost, ease of synthesis and storage, and rapid action without the need for introducing exogenous genes. They circumvent the high costs associated with cell therapies and potential side effects from allogeneic cell transplantation, holding significant promise and potential in both biomedical research and clinical applications.

Fibroblasts are a cell type with functional and transcriptional heterogeneity and cellular fate plasticity, making them a promising source of seed cells for clinical tissue and organ repair and regeneration [3]. Small molecules have become recognized as crucial tools for the induction of cellular reprogramming. In our laboratory’s previous research, a novel small chemical molecule probe, CPP ((E)-4-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl)piperazin-1-yl)styryl)-1-methylpyridin-1-ium iodide), was identified as an inhibitor of prolyl-4-hydroxylase 2 (PHD2) [4]. CPP induces the differentiation of cultured human dermal fibroblasts into vascular endothelial cells (VECs) via the PHD2/hypoxia-inducible factor-1α/hairy-related transcription factor 1 (PHD2/HIF1α/HEY1) signaling pathway [4,5]. The differentiated VECs exhibited therapeutic efficacy in treating lower limb ischemia in mice, indicating that CPP holds promise as a therapeutic candidate for limb ischemia [5]. In this study, we aimed to investigate the capability of CPP to directly induce angiogenesis in vivo under conditions of vascular injury.