(I-324) Leveraging Short Chain Fatty Acids as an Alternate Energy Source for Critical Limb Ischemia

Thursday, October 12, 2023

2:00 PM – 3:00 PM PDT

Location: Exhibit Hall - Row I - Poster # 324

Presenting Author(s)

NJ

Nikita W. John (she/her/hers)

PhD Student
Northwestern University
Evanston, Illinois, United States

Co-Author(s)

CC

Calvin Chao

Vascular Surgery Resident
Feinberg School of Medicine, United States
CD

Caitlyn Dang

Laboratory Technician
Northwestern University Feinberg School of Medicine, United States

Last Author(s)

KH

Karen Ho

Collaborating PI
Northwestern University Feinberg School of Medicine, United States

Primary Investigator(s)

BJ

Bin Jiang

PI
Northwestern University Feinberg School of Medicine, Northwestern University, United States

Introduction:: Atherosclerosis is a cardiovascular disease characterized by plaque formation in elastic aortas and muscular arteries. These plaques can grow and harden, leading to thrombosis or occlusion of the artery¹. Peripheral artery disease (PAD) is a manifestation of this, where plaques in the lower extremity vasculature prevent sufficient blood flow to the limb, leading to local oxygen and nutrient deficiency². This progresses to muscle degeneration, fat infiltration, and cellular metabolic dysregulation in the lower leg, and if left untreated, can lead to critical limb threatening ischemia². Current treatments, such as angioplasty and stenting or surgery, aim to improve blood flow, but do not address muscle degeneration, and are not permanent solutions for PAD³. Butyrate, a short chain fatty acid produced by gut microbes has been linked to improved skeletal muscle health⁴. Mice receiving a butyrate supplemented diet from a young age experienced less muscle loss with age, compared to mice receiving a standard diet⁵. However, systemic administration of butyrate as a therapeutic is insufficient due to immediate colonocyte metabolism and significant hepatic uptake, and any remaining will not reach the affected area due to insufficient blood flow as a result of ischemia^6,7. Chen et. al. have shown that local delivery of butyrate to ischemic heart tissue improved cardiac function significantly post-injury⁸. The hypothesis of this study is that sustained butyrate delivered locally via poly(lactic-co-glycolic) (PLGA) microspheres to ischemic tissue will allow for the sustained release of butyrate, providing ischemic muscle tissue with an alternate energy source, and prevent muscle degeneration.

Materials and Methods:: Microspheres were fabricated using a water-in-oil-in-water double emulsion process. Briefly, PLGA dissolved in dichloromethane (DCM) was vortexed with sodium butyrate in distilled water to form an emulsion, then combined with 2% poly vinyl alcohol. This emulsion was added to water, stirred to allow sufficient microsphere assembly, and lyophilized after washing. Scanning electron microscopy (SEM) imaging and size distribution via light microscopy image analysis were used to characterize the microspheres. Butyrate encapsulation efficiency was determined by dissolving the microspheres in DCM, eluting the butyrate with distilled water, and reading the absorbance of the water phase at 230 nm. Release studies were also conducted by incubating the microspheres in PBS at 37℃, collecting the supernatant, and reading the absorbance at 230 nm. In addition, a murine hindlimb ischemia model was used to assess ischemic skeletal muscle response to butyrate. The right femoral artery of male C57BL/6 mice was double ligated to induce ischemia. 24 hours after injury, intramuscular injections containing either empty microspheres, butyrate-loaded microspheres, or dissolved butyrate will be administered (n = 5 mice per treatment group). Laser Doppler imaging will be conducted weekly to assess blood perfusion, and after 4 weeks, mice will be euthanized, and muscle tissue will be collected for histological analysis, including fat infiltration, muscle fiber diameter, and fibrosis formation. Statistical analysis involved unpaired Student t-test for microsphere characterization, and one-way ANOVA will be performed for data collected from animal studies, with a 95% significance level for both.

Results, Conclusions, and Discussions::

SEM imaging in Figure 1 indicated that butyrate incorporation did not affect the outer surface of the microspheres. Initial fabrication efforts focused on optimizing stirring time for adequate assembly of microspheres. From the SEM images, 3 hour stirring duration resulted in broken microspheres with variable thickness, while 48 hour stirring duration resulted in fully formed microspheres. Size distribution analysis showed microspheres had diameter of 50.6±42.65 microns for the 48 hour stir, and 76.58±53.19 microns for the 3 hour stir, indicating a variable size range for the microspheres in both batches. Encapsulation efficiency studies indicated that microspheres stirred for 48 hours had a 54% encapsulation, while the microspheres stirred for 3 hours had at 49% encapsulation. Finally, initial findings of the drug release study showed a burst release of butyrate, and a total of 7.5% of loaded drug released over 4 weeks. This indicates that butyrate is released steadily, although formulation will be altered to allow for complete elution of the drug at 4 weeks, which can be seen in Figure 1. The hindlimb ischemia model has been established, and double ligation of the femoral artery led to a 91% decrease in blood reperfusion, muscle degeneration and fat infiltration resulting from acute ischemia (see Figure 2). Therefore, this model will be able to evaluate the effects of butyrate on ischemic muscle tissue.

Preliminary characterization studies indicate that encapsulation of butyrate within the microspheres was successful, and does not affect the formulation or surface of the microspheres. In addition, butyrate was successfully released from the microspheres, and its release was consistent over the course of four weeks. With this initial data and previous studies indicating the protective effects of butyrate on muscle, this study will elucidate butyrate’s effects on ischemic muscle tissue, its potential therapeutic application for muscle degeneration due to PAD, and potentially open the door to studying microbe-derived metabolites as therapeutic agents.

Acknowledgements (Optional): :

References (Optional): :

1. Libby, P. et al. Atherosclerosis. Nat. Rev. Dis. Primer 5, 1–18 (2019).

2. Weiss, D. J. et al. Oxidative damage and myofiber degeneration in the gastrocnemius of patients with peripheral arterial disease. J. Transl. Med. 11, 230 (2013).

3. Brass, E. & Hiatt, W. Acquired skeletal muscle metabolic myopathy in atherosclerotic peripheral arterial disease. https://journals.sagepub.com/doi/epdf/10.1177/1358836X0000500109 doi:10.1177/1358836X0000500109.

4. Han, D.-S. et al. Differences in the gut microbiome and reduced fecal butyrate in elders with low skeletal muscle mass. Clin. Nutr. 41, 1491–1500 (2022).

5. Walsh, M. E. et al. The histone deacetylase inhibitor butyrate improves metabolism and reduces muscle atrophy during aging. Aging Cell 14, 957–970 (2015).

6. Nooromid, M. et al. Microbe‐Derived Butyrate and Its Receptor, Free Fatty Acid Receptor 3, But Not Free Fatty Acid Receptor 2, Mitigate Neointimal Hyperplasia Susceptibility After Arterial Injury. J. Am. Heart Assoc. Cardiovasc. Cerebrovasc. Dis. 9, e016235 (2020).

7. Louis, P. & Flint, H. J. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol. Lett. 294, 1–8 (2009).

8. Cheng, P. et al. PLGA‐PNIPAM Microspheres Loaded with the Gastrointestinal Nutrient NaB Ameliorate Cardiac Dysfunction by Activating Sirt3 in Acute Myocardial Infarction. Adv. Sci. 3, 1600254 (2016).