Collagen type 1

EC were plated onto six-well silicone elastomer Bioflex plates coated with type I collagen (FlexCell International, Hillsborough, NC) and grown to 75–80% prior to transfection as described. Mechanical stretch was performed via the Flexcell Strain Unit (FX-3000; FlexCell International) placed in a 5% CO₂ incubator at 37 °C and 95% humidity. The device uses a controlled vacuum to induce CS with 18% elongation at a frequency of 30 cycles per minute (0.5 Hz) for 6 h. The media was then collected and briefly centrifuged to measure cytokine levels with Bio-Rad Bio-Plex ELISA kits (Hercules, CA) according to the manufacturer’s instructions.

For the LPS challenge, ECs were cultured in EGM-2 and challenged with 100 ng/ml of LPS for various time points as indicated. The resulting media were collected and briefly centrifuged using ELISA assay (Biolegend, San Diego, CA, United States) according to the manufacturer’s instructions. For CS experiments, ECs were plated onto six-well silicone elastomer Bioflex plates coated with type I collagen (FlexCell International, Hillsborough, NC, United States) and grown to 75–80% prior to transfection as described. Mechanical stretch was performed via the Flexcell Strain Unit (FX-3000; FlexCell International) placed in a 5% of CO₂ incubator at 37°C and 95% of humidity. The device uses a controlled vacuum to induce CS with 18% of elongation at a frequency of 30 cycles per minute (0.5 Hz) for 4 h. The media were then collected and briefly centrifuged prior to the ELISA assay.

MLE-12 were seeded onto six-well BioFlex plates coated with type I collagen (Flexcell), grown to confluence, and then exposed to pathological and physiological (18% and 5% elongation respectively) CS for 48 h, whereas the static plate was placed in the same incubator as the control. After 48 h, the lipids were extracted from the cells and the sphingolipid levels were measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The expression of sphingolipid metabolizing enzymes and apoptotic markers were identified in the cell lysates using western blotting. Prior to subjecting the cells to CS, they were pretreated with 4-DP to inhibit S1P Lyase activity in the cells. Flow cytometry was used to identify the late apoptotic cells using FITC-Annexin V and PI staining. CS- induced gap formation between the cells was studied by staining the cells using F-actin and E-Cadherin. The secretion of cytokines was measured from the supernatants using ELISA. Densitometry analysis and gap formation computation was performed using ImageJ software (https://imagej.nih.gov/ij/). The date here was part of a work of the thesis of Vidyani Suryadevara for her Masters in Science [72 ]. Detailed methods have been stated in the Supplementary methods section of this manuscript.

Cells were passaged to DMEM containing 10% FBS six-well BioFlex plates coated with collagen type I (Flexcell) for 24 h to form a confluent monolayer. Then the medium was replaced by DMEM without FBS for 6 h before stretch. A Flexcell FX-5000TM Tension System (Flexcell International Corporation, Burlington, CA, USA) was used to order cyclic mechanical stretch. Cells were stretched at a frequency of 1HZ with 20% amplitude and a 1:1 stretch:relaxation ratio for 15 min, 2 h, 6 h, 12 h and 24 h. Both the tension system and the control plates were placed in the incubator at 37 °C, with 5% CO₂.

BMSCs were plated at a density of 5 × 10⁵ cells/cm² (if not mentioned) in 1 ml of medium on six-well flexible silicone rubber BioFlex plates coated with collagen type I (Flexcell International Corporation, Hillsborough, NC, USA). Cells were cultured for 24 h to reach 50–60% confluency before mechanical tension was applied, which guaranteed sufficient space for cell proliferation and an adequate number of cells for the following experiments. Cyclic mechanical stretch (CMS) with a 0.5-Hz sinusoidal curve at 10% elongation was applied using an FX-5000 T Flexercell Tension Plus unit (Flexcell International Corporation). The cultures were incubated in a humidified atmosphere at 37 °C and 5% CO₂ during the stretching. Cells were harvested immediately after the application of CMS stimulation was completed. Control cells were cultured on the same plates in the same incubator but were not subjected to stretching.