Using single cell RNA-seq analysis, researchers showed that TGFβ induced inflammation in endothelial cells in mouse models. 

In a new Yale-led study, investigators have revealed previously unknown factors that contribute to the hardening of arteries and plaque growth, which cause heart disease. Their insight is the basis for a promising therapeutic approach to halt and potentially reverse plaque buildup and the progression of disease, the researchers said.

Current treatments for plaque and hardened arteries, a condition known as atherosclerosis, can slow but not improve the disease. Experts believe that may be due to ongoing inflammation in blood vessels. To understand the factors contributing to this inflammation, the research team focused on a group of proteins, called transforming growth factor beta (TGFß), that regulates a wide range of cells and tissues throughout the body.

Using cultured human cells, the researchers discovered that TGFβ proteins trigger inflammation in endothelial cells — the cells that form the inner lining of artery walls — but not in other cell types. With a technique called single cell RNA-seq analysis, which measures the expression of every gene in single cells, they then showed that TGFβ induced inflammation in these cells in mouse models. This finding was notable, said the researchers, because TGFβ proteins are known to decrease inflammation in other cells in the body.

The researchers also showed that when the TGFβ receptor gene is deleted in endothelial cells, both the inflammation and plaque in blood vessels are significantly reduced.

To test this approach as a potential therapy, the team, led by professor of medicine Michael Simons, M.D., used an “interfering” RNA, or RNAi, drug developed at Yale, to disrupt TGFß receptors. Interfering RNA use a gene’s own DNA sequence to turn off or silence the gene. To deliver the drug only to endothelial cells in the blood vessel walls of mice, they employed microscopic particles, or nanoparticles, created by their co-authors at MIT. This strategy reduced inflammation and plaque as effectively as the genetic technique.

TGF-β endothelial-specific induction of inflammatory response

a,b, Bulk RNA-seq analysis of HUVEC (EC), HASMC (SMC), CD4⁺ effector memory T cell (T_EM) and macrophage (M) gene expression before and after stimulation with TGF-β1 (a) and inflammatory gene expression (b). Each lane is an average of two biologically independent samples. c, ChIP-seq analysis of canonical TGF-β signalling in ECs and SMCs. Note increased SMAD2/3 binding to regulatory elements of CCL2, CLDN5 and SERPINE1 in ECs but not SMCs. Pol II S2 and Pol II S5 ChIP in combination with bulk RNA-seq indicate increased expression of CCL2 and SERPINE1 and decreased expression of CLDN5 after TGF-β1 stimulation; n = 1 per experimental condition. d, Relative CLDN5 expression in primary ECs isolated from control (Cdh5-CreER^T2;Tgfbr1^fl/fl;Tgfbr2^fl/fl;mT/mG mice treated with corn oil) and TGF-βR^iEC (Cdh5-CreER^T2;Tgfbr1^fl/fl;Tgfbr2^fl/fl;mT/mG mice treated with tamoxifen) mice. Bars throughout indicate the mean ± s.e.m. 0 h, P = 0.004; 6 h, P = 0.001. Two-tailed Student’s t-tests (n = 3 animals per group for 0 h, n = 6 animals per group for 6 h). e, Intravenous Evans blue dye administration following a subcutaneous TNF-α injection into mouse ear. Scale bar, 2 mm; 0 min, P = 0.56; 15 min, P = 0.000066, two-tailed Student’s t-tests (n = 6 animals per group for 0 min, n = 7 animals per group for 15 min). f,g, Mouse ear sections were stained with anti-ICAM, VCAM-1, Ly6G and CD45 antibodies 6 h and 3 days following a subcutaneous TNF-α injection. Note a strong reduction in expression of both adhesion molecules (f) and reduced presence of inflammatory cells (g) in TGF-βR^iEC mice. Scale bar, 16 μm; n = 7 animals per group. ICAM1⁺ area 6 h, P = 0.001; ICAM1⁺ area 3 d, P = 0.0002; VCAM-1⁺ area 6 h, P = 0.0001; VCAM-1⁺ area 3 d, P = 0.00001; Ly6G⁺ cells 6 h, P = 0.0499; Ly6G⁺ cells 3 d, P = 0.0004; CD45⁺ cells 6 h, P = 0.051; CD45⁺ cells 3 d, P = 0.00004. Two-tailed Student’s t-tests.

The findings identify TGFß signaling as a major cause of chronic vessel wall inflammation, and demonstrate that disruption of this pathway leads to cessation of inflammation and substantial regression of existing plaque, said the scientists.

Based on this discovery, investigators at Yale and MIT have launched a biotech company, VasoRX, Inc., to develop this targeted approach, using the RNAi drug delivered by nanoparticles as a potential therapy for atherosclerosis in people.

Source – Yale University