PGI2 pathway to enhance wound healing in diabetic foot ulcers – PGI2Heal

We will first explore the alterations of the PGI2 pathway in the skin of mouse models of diabetes-related ulcers. Pharmacological modulation of PGI2-dependent microvascular reactivity; and gene and protein expression of key components of the PGI2 pathway, pro- and anti-inflammatory mediators, and angiogenesis; will be compared between diabetic mice and controls. Then, we will decipher the pathways involved using knockout mice. Tissue specific inactivation using CRISPR-Cas9 technique will finally confirm the results. Then, we will explore the PGI2 pathway in diabetic patients with and without DFU. Cutaneous perfusion will be assessed with laser speckle contrast imaging while stimulating or inhibiting the PGI2 pathway using intradermal microdialysis. Skin biopsies will also be collected to quantify cutaneous expression (gene and protein) of key components of the PGI2 pathway, and for subsequent in vitro experiments. Indeed, we will then decipher the mechanisms underlying the involvement of the PGI2 pathway on cell migration, angiogenesis and tissue remodeling in vitro. Finally, innovative 3D models of reconstructed skin containing microvessels will be used to further explore PGI2-dependent abnormalities in models of diabetic skin.

The first results obtained on cutaneous gene expression show abnormalities in STZ diabetic mice. In addition,
we evaluated microvascular reactivity assessed by current-induced vasodilatation (CIV), which stimulates C nerve fibers and prostaglandin production. Despite a moderate CIV in healthy mice, this was significantly reduced after blocking the COX pathway with indomethacin (ip 5mg/kg). Association with a NO synthase blocker (LNNA ip 20mg/kg) does not alter the response. In STZ mice, CIV is significantly altered suggesting an altered PGI2-dependent response. The effect of blocking the PGI2 pathway and other signalling pathways of the endothelial response, as well as the response to PGI2-independent stimuli, is being explored in these mice. Similarly, we are currently evaluating this response in Ptgir-/- mice.
Microvascular dysfunction in diabetic mice is associated with delayed wound healing, which we have observed in STZ and db/db mice. In db/db mice, results show that the administration of a PGI2 analogue by iontophoresis, but not current alone, is associated with accelerated re-epithelialization. The pharmacokinetic study shows that this effect is not systemic, but localized to the site of application. We also have a non-significant tendency to an improvement in endothelium-dependent reactivity after treatment.
Finally, we have developed a vascularized reconstructed skin model. For this purpose, fibroblasts and human endothelial cells (HUVEC or HDMECS) were seeded in a cross-linked fibrin hydrogel, then healthy donor keratinocytes were seeded to form the epidermis. We obtain a dermis rich in extracellular matrix and a differentiated epidermis with vascular structures.

Analyses of gene and protein expression are continuing to better understand the mechanisms explaining the observed differences between groups. For the remainder of the project, pro and anti-inflammatory mediators, markers of angiogenesis and vascular remodeling will be evaluated in pre- and post-healing mouse models. Also in animals, we will explore the mechanical and structural properties of healed skin after iontophoresis of a PGI2 analogue in diabetic mice.
We also aim to evaluate PGI2-dependent cutaneous microvascular function in humans by comparing reactivity using laser speckle contrast imaging coupled with dermal microdialysis. The study is based on the inclusion of three groups of volunteers: healthy subjects, diabetic patients without foot ulcers, and diabetic patients with foot ulcers.
In addition, skin biopsies will be taken in order to produce pathological 3D reconstructed skins, using fibroblasts from the patients included in this study. We obtained a favorable opinion from the French Ethics Committee on September 08, 2020 and from the ANSM on September 18, 2020. Inclusions should start at the end of 2020.

This research program has resulted in two original manuscripts (1 multi-partner, and 1 single-partner) being finalized, as well as 3 oral communications.
There is also a patent registered in France: RACHIDI W, JOBEILI L, LELLOUCH, A and LANTIERI L. French application N/Ref: B04325 FR - G157-B2019-16, March 30, 2020: Biomaterial comprising a resorbable porous matrix and associated manufacturing process.
As well as a new collaborative project: two of the partners of this project (LBTI, CHUGA) are partners of a second ANR project (AAPG 2019) led by the University of Avignon.

Diabetic foot ulcers (DFUs) are a common and serious complication of diabetes, and associated with important morbidity. Despite available treatments, they are responsible for a lower limb loss every 30 seconds somewhere in the world. The skin microcirculation, by delivering oxygen and nutrients, has a key role in tissue survival. Structural and functional abnormalities of the cutaneous microcirculation in diabetic patients with ulcer support its critical role in the pathophysiology of DFUs. However, the detailed mechanisms underlying such dysfunction remain largely unexplored. The prostacyclin (PGI2) is an important regulator of vascular homeostasis and a potent vasodilator produced by endothelial cells. It also plays a role in angiogenesis, and regulates fibroblast and keratinocyte migration and proliferation. We hypothesize that impaired PGI2 pathway in the skin is involved in the pathophysiology of DFUs, and that targeting the PGI2 pathway may provide a treatment for DFUs.
Although intravenous PGI2 analogs have been used for decades to treat other types of ulcers, they expose the patient to dose-limiting side effects, with safety issues and increased costs. The paradox is that impaired microvasculature prevents the drug from diffusing properly to the wound. Local administration may limit the toxicity of PGI2 analogs and decrease costs. Iontophoresis is a drug delivery method based on the transfer of ions using a low-intensity electric current. Interestingly, electric stimulation also increases PGI2 release in the skin, leading to vasodilation. It may also have a positive impact on wound healing by increasing the migration of keratinocytes, fibroblasts and neutrophils. We therefore hypothesize that using low-intensity current both as a vector for local delivery of a PGI2 analog (iontophoresis), and for its proper effect on wound healing, potentiate the stimulation of the PGI2 pathway and improves healing.
To address these questions, we will conduct closely connected studies in animals, humans, and 3D in vitro models. We will first explore the alterations of the PGI2 pathway in the skin of mouse models of diabetes-related ulcers. Pharmacological modulation of PGI2-dependent microvascular reactivity; and gene and protein expression of key components of the PGI2 pathway, pro- and anti-inflammatory mediators, and angiogenesis; will be compared between diabetic mice and controls. Two types of wounds will be performed (excisional and ischemic). Then, we will decipher the pathways involved using knockout mice. Tissue specific inactivation using CRISPR-Cas9 technique will finally confirm the results. Then, we will explore the PGI2 pathway in diabetic patients with and without DFU. Cutaneous perfusion will be assessed with laser speckle contrast imaging while stimulating or inhibiting the PGI2 pathway using intradermal microdialysis. Skin biopsies will also be collected to quantify cutaneous expression (gene and protein) of key components of the PGI2 pathway, and for subsequent in vitro experiments. Indeed, we will then decipher the mechanisms underlying the involvement of the PGI2 pathway on cell migration, angiogenesis and tissue remodeling in vitro. Innovative 3D models of bio-printed skin containing microvessels will be used, to explore in depth the interaction between low-intensity current and the PGI2 pathway. Finally, we will assess the effect of iontophoresis of a PGI2 analog on wound healing in diabetic mice. It will be compared to current alone and to sham procedure (dressing only), to test whether PGI2 and electric current potentiate in vivo. While most studies focus on the time to healing, we will also explore the biomechanical properties and structural modifications of healed skin. We hypothesize that besides its effects on functional microvascular reactivity, the treatment influences deeper tissue remodelling. Such effects would have some bearing on the risk of relapse, which is a hallmark of chronic wounds in humans.