# Short-Chain Fatty Acids and Palmitate Induce Distinct Metabolic and Phenotypic Signatures in Normal and Ischemic Skeletal Muscle Microvascular Endothelial Cells

## TL;DR

Short-chain fatty acids improve blood vessel growth in ischemic muscle cells, while palmitic acid worsens their function, suggesting potential treatments for peripheral artery disease.

## Contribution

The study reveals distinct metabolic and functional effects of short-chain fatty acids and palmitate on ischemic endothelial cells.

## Key findings

- Palmitate reduces endothelial cell survival, angiogenic capacity, and barrier integrity in ischemic conditions.
- Short-chain fatty acids enhance glycolysis–mitochondrial OxPhos coupling and angiogenic capacity in ischemic endothelial cells.
- SCFAs improve barrier integrity and metabolic health, unlike palmitate, which impairs it.

## Abstract

What are the main findings?
Palmitic acid aggravates ischemic vascular dysfunction by decreasing endothelial cell survival, angiogenic capacity, barrier integrity, and overall metabolic health.Short-chain fatty acids enhance glycolysis–OxPhos coupling in ischemic endothelial cells to induce their angiogenic capacity with preserved barrier integrity.

Palmitic acid aggravates ischemic vascular dysfunction by decreasing endothelial cell survival, angiogenic capacity, barrier integrity, and overall metabolic health.

Short-chain fatty acids enhance glycolysis–OxPhos coupling in ischemic endothelial cells to induce their angiogenic capacity with preserved barrier integrity.

What are the implications of the main findings?
Palmitic acid could be a contributing factor to the disease severity observed in peripheral artery disease patients with diabetes.Short-chain fatty acids could be a potential therapeutic used to revascularize ischemic muscle in patients suffering from peripheral artery disease.

Palmitic acid could be a contributing factor to the disease severity observed in peripheral artery disease patients with diabetes.

Short-chain fatty acids could be a potential therapeutic used to revascularize ischemic muscle in patients suffering from peripheral artery disease.

Background: Palmitate, a long-chain fatty acid, is well known to be a significant risk factor for cardiovascular diseases. In our current study, we wanted to determine whether palmitate treatment further aggravates ischemic endothelial cell (EC) injury and can serve as an in vitro model that emulates diabetic peripheral artery disease (diabetic-PAD). Short-chain fatty acid (SCFA) treatment was used as an additional comparator for palmitate-induced vascular dysfunction in normal or ischemic ECs in vitro. Methods: Hypoxia serum starvation (HSS) was used as an in vitro model for PAD. Cell survival or proliferation was determined by the CCK8 kit. EC angiogenic capacity was determined by in vitro tube formation assays on growth factor-reduced Matrigel. EC barrier integrity was determined by trans-endothelial electrical resistance measurements by EVOM3. EC metabolic phenotyping was performed by Seahorse glycolysis, mitochondrial respiration, and fatty acid oxidation metabolic assays. Results: Palmitate dramatically decreased the survival of normal and ischemic ECs, whereas SCFAs did not have a significant effect on ischemic EC survival. In vitro angiogenic assays showed that palmitate significantly decreased the angiogenic capacity of ischemic ECs, whereas SCFAs significantly induced their angiogenic capacity. While palmitate significantly decreased normal and ischemic EC barrier integrity, SCFAs improved normal and ischemic EC barrier integrity. Metabolic assays showed that palmitate significantly decreased normal EC mitochondrial respiration but not glycolysis. However, palmitate significantly decreased overall metabolic health, including mitochondrial respiration and glycolysis in ischemic ECs. On the contrary, SCFAs increased both mitochondrial respiration and glycolysis in normal ECs. In ischemic ECs, SCFAs induced mitochondrial respiration with a concomitant decrease in glycolysis. Fatty acid oxidation analysis showed that, unlike palmitate, which depends on carnitine palmitoyl transferases (CPTs) for β-oxidation in both normal and HSS ECs, SCFAs depend partly on CPTs to undergo β-oxidation in HSS ECs but not in normal ECs. Conclusions: While palmitate inhibits ischemic EC angiogenic capacity by decreasing overall metabolic health, SCFAs induce glycolysis–mitochondria OxPhos coupling to induce ischemic EC angiogenic capacity.