Abstract Tu0084: Cilia-dependent Hedgehog Signaling Act as A Guardian against Limb Ischemia-induced Fatty Fibrosis

Chronic limb-threatening ischemia (CLTI) is the most severe form of peripheral arterial disease (PAD). Only 40% (2 of 5) of CLTI patients benefit from the current treatments, highlighting the need for alternative strategies. The replacement of skeletal muscle with intramuscular adipose and fibrotic tissue, generated from fibro/adipogenic progenitors (FAPs), is a hallmark of CLTI as it signals the transition from PAD to CLTI. We previously discovered that FAPs possess a primary cilium, which receives and interprets extracellular cues, such as Hedgehog signaling. Once activated upon injury, Hedgehog acts as adipogenic brake and represses intramuscular fat formation, while at the same time promotes regenerative myogenesis. Intriguingly, our preliminary data indicate that Hedgehog signaling is being disrupted in CLTI patients, thereby providing the exciting hypothesis that ciliary Hedgehog signaling acts as adipogenic brake in PAD/CLTI patients and that loss of this brake allows the formation of intramuscular fat and accelerates the decline in muscular and vascular health. Through immunofluorescence, I found that femoral artery ligation-induced hind limb ischemic (HLI) injury rapidly induced FAPs to ciliate, indicating that cilia are present at the right time and place to control FAP fate and function during HLI. While present, cilia-dependent Hedgehog signaling is lost in PAD/CLTI patients, indicating that re-activation could be beneficial. I then used pharmacological and genetic approaches to activate Hedgehog in our ischemia-susceptible BALB/c mouse model (N=6-8/group). Excitingly, both the treatment with a small molecule Hedgehog pathway activator Smoothened agonist (SAG-21K) and genetically removing cilia specifically from FAPs using a tamoxifen-inducible FAP-specific Cre allele ( Pdgfrα CreERT x Ift88 lox/lox ) lead to Hedgehog activation according to Hedgehog target Gli1 expression measured via RT-qPCR. Importantly, Hedgehog re-activation strongly suppressed intramuscular fat accumulation (Control: 222.54 ± 126.14 adipocytes/mm^2; Treatment: 65.98 ± 41.49 adipocytes/mm^2), and enhanced perfused vascular density (Control: 122.83 ± 65.09 capillaries/mm^2; Knockout: 250.38 ± 75.41 capillaries/mm^2). Together, my data demonstrate that re-activation of ciliary Hedgehog signaling can alleviate PAD/CLTI pathology, thereby providing an innovative therapeutic approach.