Prospective, monocentric, pathophysiological study, comparing 3 parallel groups: healthy controls; patients with diabetes and without DFU; patients with diabetes and with DFU. To address secondary objectives, samples from a fourth group will be collected.

Diabetic foot ulcers (DFUs) are a common and serious complication of diabetes mellitus, and associated with major morbidity. Indeed, diabetes is the primary cause of non-traumatic lower-limb amputation, and the rise in the prevalence of type 2 diabetes worldwide increases the global burden of DFUs. The treatment of DFUs is particularly challenging. Besides etiologic measures, local therapy of foot ulcers mainly relies on debridement of the wound and dressings. Essential complementary measures include pressure off-loading and infection control. However, despite these treatments, complications are frequent, stressing the need for new treatments.

The microcirculation has a key role in tissue survival, and several classical pathways explain how hyperglycemia damages the microvessels. There is growing evidence that the PGI2 pathway is dysregulated in diabetes, which contributes to microvascular dysfunction. Besides its vasodilator effect, recent data has revealed the major role of PGI2 in angiogenesis. In the skin, such effect on healing might be enhanced by the role of PGI2 in the regulation of fibroblast and keratinocytes migration and proliferation.

In the past few decades, studies in diabetic patients with ulcers have shown numerous structural and functional abnormalities of the cutaneous microcirculation, supporting its critical role in the pathophysiology of DFUs. However, the detailed mechanisms underlying endothelial dysfunction in the skin of diabetic patients remain largely unexplored in vivo. A better understanding of the specificities of microvascular changes in the diabetic foot is essential to developing new treatments for this pressing clinical need.

Objectives are

• to explore the role of the PGI2 pathway in skin microvascular reactivity, in healthy subjects and in diabetic patients with and without DFU
• To determine the involvement of COX-1 and COX-2 in cutaneous current-induced vasodilation (CIV), in healthy subjects and in diabetic patients with and without DFU.
• To determine the involvement of sensory nerves in cutaneous CIV, in healthy subjects and in diabetic patients with and without DFU.
• To compare the function of the IP receptor between healthy subjects and diabetic patients with and without DFU.
• To determine the involvement of the nitric oxide (NO) and epoxyeicosatrienoic acids (EETs) pathway in cutaneous CIV, in healthy subjects and in diabetic patients with and without DFU.
• To assess cutaneous expression of the different components of the PGI2 pathway in the skin of healthy subjects and of diabetic patients with and without DFU.
• To assess the role of the PGI2 pathway on cell migration in vitro

Inclusion criteria:

Groups 1,2,3,4:

• Informed consent signed
• Affiliated to social security insurance or beneficiary of social security insurance
• Aged of 18 or older

Group 1: healthy volunteers:

-Free from all acute and chronic pathology

Group 2: diabetic patients without DFU:

-Patients with type 2 diabetes according to the criteria of the American Diabetes Association (ADA), without DFU or history of DFU

Group 3: diabetic patients with DFU or recent history of DFU (occurred within the last two years):

-Patients with type 2 diabetes according to the criteria of the American Diabetes Association (ADA) with: One or more active grade 1A, 1C, 2A or 2C (University of Texas Classification of Diabetic Foot) foot ulcer of microvascular or mixed etiology; Or a recent history (<2 years) of foot ulcer of microvascular or mixed etiology.

Group 4 (to collect samples of foot skin biopsies to address secondary objectives ):

-Patients with type 2 diabetes according to the criteria of the American Diabetes Association (ADA),with neuropathy and DFU undergoing lower-limb surgery for skin ulcer (e.g. toe amputation).

Exclusion criteria

Groups 1, 2 and 3:

• Unstable diabetes that has resulted in hyperosmolar coma or ketoacidosis, and/or documented increase or decrease in HbA1c of more than 2.0% within the previous 3 months.
• Presence of diabetic peripheral neuropathy above the ankle, defined as a scoring >3 of at least two of the four stimuli of the Neuropathy Disability Score (i.e. pinprick sensation, light touch, vibration, and temperature perception) (37).
• Infected wound, treated with antibiotics in the past 15 days.
• Critical ischemia of the lower limb, defined as leg pain at rest associated with ankle pressure <70 mmHg.
• History of hypersensitivity reaction to treprostinil, fluconazole, other azole compounds, L-NMMA, ketorolac, meloxicam, or any NSAIDs or acetylsalicylic acid, lidocaine (or any local anesthetic with an amide bond), or their excipients
• History of asthma, rhinitis, nasal polyps, angioedema, hives rash, or any other allergic reaction due to acetylsalicylic acid or any NSAID taking
• Pulmonary veno-occlusive disease (PVOD)
• Porphyria
• Hyperkalemia
• Active or uncontrolled cardiovascular disease as follows: Myocardial infarction, or angina within the previous 6 months; Severe ischemic heart disease; Arrhythmia (uncontrolled, symptomatic, requiring treatment or life-threatening); Congestive heart failure, or decompensated heart failure not medically controlled; Stroke or transient ischemic attack within the previous 3 months; Uncontrolled hypertension: systolic blood pressure (SBP)> 180 mmHg or diastolic blood pressure (DBP)> 105 mmHg (2 abnormal readings during visit) Valvular heart disease
• Severe liver disease (Child-Pugh C) at the time of enrollment
• Renal disease (creatinine >2 mg/dL and/or estimated glomerular filtration rate (GFR) <30 mL/min, history of dialysis)
• Active peptic ulcer disease, recent gastrointestinal bleeding or perforation, or history of peptic ulcer disease or gastrointestinal bleeding or perforation with NSAIDs
• Intracerebral or gastrointestinal hemorrhage, hemostasis disorder or every clinical status that may lead to bleeding
• Chronic venous disease defined as stage 4a and 4b of the Clinical-Etiological-Anatomical-Pathophysiological (CEAP) classification
• Cutaneous condition deemed incompatible with skin biopsy and dermal microdialysis by the investigator
• History of necrotic angiodermatitis
• Trauma or any clinical event susceptible to be responsible for hemorrhage within the previous 6 months
• Concomitant treatment with pentoxifylline, anticoagulants, probenecid, medicinal products known to prolong the QT interval or anti-inflammatory or analgesic dose of acetylsalicylic acid
• If concomitant treatment with NSAIDs, participants have to be stopped 1 week before the inclusion
• Pregnancy or Lactation
• Females with childbearing potential, defined as a premenopausal female capable of becoming pregnant, and not using an highly effective form of birth control. Effective birth control methods include: oral, implant or patch hormone contraception; intrauterine device; abstinence and outercourse; tubal ligation; vasectomy.

Groups 1,2,3 and 4:

• Participant involved in another interventional clinical study
• Person deprived of liberty by judicial order
• Person under guardianship or curatorship

Qualified researchers can request access to anonymized individual patient-level data sending an e-mail to : jlcracowski@chu-grenoble.fr or mroustit@chu-grenoble.fr