Research

Another Productive Year in the Falsetta Lab

By: Megan L. Falsetta (Wood), PhD Assistant Professor

The Falsetta lab had a productive year exploring the mechanisms of vulvar disease with the goal of identifying new targets for improved therapeutics. The majority of the work in the lab focuses on localized provoked vulvodynia (LPV), chronic vulvar pain that is localized to the ring of tissue immediately surrounding the vaginal opening known as the vestibule. Their research, supported by NIH R01 HD092334 entitled “Mechanisms of vulvodynia involving dysregulation of pro-resolving lipids” carries on the long legacy of vulvodynia research at URMC. The causes of LPV are poorly understood; the Falsetta team has uncovered inflammatory mechanisms that contribute to vulvar pain and may be a root cause of vulvodynia. In a seminal publication in the Journal of Pain this past Fall1, they demonstrated that by targeting these inflammatory mechanisms using natural products (fish oil) or synthesized versions of natural products (maresin 1) they were able to completely ameliorate signs of pain in their new mouse model of vulvodynia. This model recapitulates the key aspects of vulvodynia, where chronic exposure to yeast results in sustained sensitivity, even after acute inflammation is resolved. Work is ongoing to improve the ergonomics and data collection in the model. In collaboration with Dr. Ronald Wood, PhD (OB/GYN Research), the group has developed a co-bot to one day automate threshold testing. This model includes blinding, video recording of mouse behaviors, contingent delivery of a preferred solution to maintain mouse position, electronic von Frey

measurement with custom software that automatically records peak force values, simultaneous non-invasive measurement of inflammatory markers in vulvovaginal fluid, and periodic evaluation of mouse behavioral conditioning. An ergonomic XBOX-controller run system with 4 stepping motors eliminates the fatigue associated with chasing the mouse and reduces variability, while increasing sensitivity. The Falsetta lab recently found that sustained inflammation goes hand in hand with deficits in inflammatory resolution. Patients with vulvodynia have reduced levels of pro-resolving lipids at painful sites, which may help to explain the chronicity of inflammation. They used targeted LC MS performed by their longtime collaborators at Wayne State University, Drs. Kenneth Honn, PhD and Krishna Rao Maddipati, PhD to assess nearly 200 lipid profiles. Dr. Tanzy Love, PhD (Biostatistics) performed statistical analysis to parse out the key differences. Hypersensitivity to inflammatory stimuli in combination with reduced pro-resolving mediator production results in the “perfect storm” that perpetuates inflammation leading to pain and loss of function. These lipid profiles represent a “vulvodynia signature” where certain pro-resolving lipids are deficient, while proinflammatory lipids are abundant. This signature could one day be used to diagnose patients and assess disease progression or response to treatment. It would be an ideal new primary outcome for clinical trials; it is quantitative and not subjective like current trial measures that rely upon patient perceptions of their pain (e.g. visual analog scales).

This signature also offers several new targets for therapeutic intervention, including 3 major enzymes families involved in the production of pro-resolving and proinflammatory mediators: cytochrome P450 (CYP450), lipoxygenase (LOX), and cyclooxygenase (COX). NIH PREP student Bianca Flores is investigating CYP450, while undergraduate Biochemistry honors student Sarah Fischer is investigating the LOX and COX pathways. Existing therapies might target these pathways in an “off-label” fashion. The most promising of which, CYP450, could be targeted with vaginal yeast medications (azoles), whether over-the-counter or prescription.

However, it has been unclear how hypersensitivity to inflammatory stimuli in the vulvar vestibule induces pain. Proinflammatory mediators are associated with pain in vulvodynia and can serve as surrogate measures of pain, but it is unlikely they directly elicit pain, although some of these mediators (e.g. prostaglandin E2) have been shown to sensitize neurons. The Falsetta team recently uncovered a connection between inflammation and transient receptor potential vanilloid-type 4 (TRPV4) signaling, which is in part a result of a collaboration with Dr. David Yule, PhD (Pharmacology and Physiology). TRPV4 is ubiquitous and has been implicated in mechanical allodynia (pain upon touch) in several pain conditions. Work from technician Tamari Bekauri has demonstrated inflammation is both necessary and sufficient to initiate TRPV4 signaling in a fibroblast model of vulvodynia. This signaling may create the action potential necessary to initiate pain signaling in the surrounding nerves. Previous studies have found that nerve density is increased in the vestibular tissue of vulvodynia patients, and many refer to this type of vulvodynia as neuroproliferative vulvodynia. The Falsetta lab findings support this and suggest vulvodynia is likely a neuroinflammatory disease. This is a relatively new area of exploration for the lab group and is the subject of a recent R01 application (HD108173) entitled “Transient Vanilloid Receptors and Vulvar Pain: New Therapeutic

Targets for Vulvodynia,” which scored 15th percentile upon its first submission. Furthermore, maresin appears to target this pathway in addition to its pro-resolving effects, which may make it an ideal therapeutic for vulvodynia. It is natural and safe and appears to act on more than one branch of the vulvodynia mechanism. The Falsetta lab collaborates with two pharmaceutical groups and is moving towards clinical implementation. They are planning a multicenter clinical trial in partnership with and SPM Therapeutics Inc. and former URMC physician Adrienne Bonham, MD (Oregon Health and Sciences University), which should start enrolling subjects by 2023 to test the ability of maresin to alleviate vulvar pain. Dr. David Foster, MD (OB/GYN Emeritus) is also vital in this project, drawing on his vast clinical trial expertise. In addition, the Falsetta team is working with Serentrix, Inc. on the role of TRPV channels in vulvar pain signaling and submitted a STTR entitled “Novel site specific non-opioid treatment for vulvodynia” in January of 2022 with contributions from undergraduate Biochemistry student Adam Hopson. The Falsetta lab will conduct the preclinical trials of Serentrix’s patented TRPV antagonist in their mouse model of vulvodynia. If it produces analgesic effects in mice, phase 2 funding would support a clinical trial.

The group also has pilot funding from the Mae Stone Goode Foundation entitled “The Role of Scarring in Vulvar Lichenoid Disease: New Paths to Improving Treatment” to study vulvar lichens sclerosis (vulvar scarring). Lichens sclerosis is a common dermatological condition that can affect any body site. This work has been led by undergraduate Biochemistry student Hawraa Ahmed who found that scarring markers are elevated in lichens sclerosis affected tissues and are inducible in response to endogenous scarring signals, such as transforming growth factor beta (TGFβ). During this pilot, the research team has also established the cell strains and tools necessary to collect the remaining pieces of supporting data for a new R01, likely by the end of this academic year.

The ultimate goal is to translate basic research findings from “bench to bedside.” Dr. Falsetta is passionate about understanding communication at its most basic cellular and chemical levels. However, she recognizes that the application of this knowledge is most important. Working closely with the URMC OB/GYN clinical team, especially Dr. Mitchell Linder, MD, has helped the lab team to better understand the ways they can apply their findings in the short and long term. The group is also active in several vulvar and women’s sexual health societies (e.g. ISSVD, ISSWSH) and is looking forward to presenting at the annual ISSWSH meeting in March.

Bianca Flores was selected for a poster, and Dr. Falsetta will be presenting a podium talk. This year also marks an important transition as Dr. Constantine Haidaris, PhD (Microbiology and Immunology) retires and steps down from his position as MPI on R01 HD092334. Dr. Linder will be moving into this position, further cementing his in role as a clinical leader in vulvodynia research, carrying on Dr. Foster’s mantle.