The role of Nerve-fibroblast communication in EDS pain

Classic Ehlers Danlos Syndrome (cEDS) is characterized by skin hyperextensibility and fragility, wound healing problems, and joint hypermobility. cEDS is caused by genetic defects in genes that encode type V collagen (COL5A1 and COL5A2). Chronic widespread pain is one of the most common and intractable, yet

barely studied, complaints in all EDS patients, and cEDS patients are in great need of new non-opioid pain therapies. Research into the characteristics and mechanisms of pain in EDS is exceedingly limited. A unique collaboration between the Chicago Center on Musculoskeletal Pain (C-COMP) and the Center for Medical

Genetics at Ghent University (Belgium) has uncovered critical, novel observations in cEDS patients and in Col5a1 haploinsufficient (Col5a1+/-) mice, a validated model of cEDS. Quantitative sensory testing in cEDS patients revealed mechanical pain hypersensitivity. Likewise, Col5a1+/- mice manifest widespread mechanical

allodynia, accompanied by abnormal skin innervation by nociceptors. Fibroblasts are the principal source of abnormal type V collagen, and cEDS fibroblasts show transcriptome-wide changes, including genes related to cell proliferation, migration, wound healing, and axonal guidance. While it has been recognized that fibroblasts

may contribute to peripheral sensitization of nociceptors through secretion of sensitizing molecules, in depth studies into how fibroblasts communicate with the nociceptive system in peripheral tissues to initiate and/or potentiate pain are surprisingly scant. Here, we will address this critical knowledge gap; we propose that cEDS

provides an excellent model for investigating how fibroblast-nociceptor interactions drive pain in cEDS and other pain syndromes. We hypothesize that fibroblast-neuron interactions may cause hyperexcitability and/or abnormal growth of nociceptors in the skin, two of the defining features of cEDS. We are in an unparalleled

position to examine this idea, owing to the unique, complementary resources and state-of-the-art techniques present in our groups. In Aim 1, we will determine the innervation and cellular transcriptome in skin tissues of cEDS vs control patients through immunohistochemistry and Visium. To do this, we will leverage the unique

cohort of molecularly and phenotypically characterized cEDS patients available through the Center for Medical Genetics in Ghent, Belgium. In Aim 2, we will use a neuron-fibroblast microfluidic co-culture system to assess the effect of Col5a1 mutant fibroblasts on neuronal excitability and neuronal growth/sprouting. In Subaim 2.1,

we will use murine cells to examine the effects of fibroblasts harboring a type V collagen defect on DRG neurons in co-cultures. In Subaim 2.2, findings will be validated in human microfluidic co-cultures, using human iPSC- derived sensory neurons with fibroblasts derived from cEDS patients or controls. We anticipate that the findings

generated from the proposed experiments will advance our knowledge of mechanisms underlying pain in cEDS, and will generate important new information into mechanisms of more prevalent painful conditions where neuron- fibroblast interactions play a role in the production of abnormal skin innervation and pain.