Antibody based functional blocking of the ED-A domain of Fibronectin reduces expression of inflammatory genes in murine lungs in Sugen5416/Hypoxia induced pulmonary hypertension

Background: Inflammation and subsequent pulmonary vascular remodelling are pivotal processes in the development of pulmonary hypertension (PH). Our recent research delved into an innovative therapeutic approach that involved the application of a human recombinant antibody specifically targeting and functionally blocking the extra domain A of the extracellular matrix protein Fibronectin. This particular domain is not expressed in healthy adult tissues but displays significant transcriptional activation in PH. Our findings revealed encouraging outcomes in terms of mitigating the progression of the disease, a result not observed in the control group that received sham treatment.

We hypothesized that an @ED-A⁺ Fn antibody treatment possesses immunomodulatory properties, creating a less inflammatory environment. To investigate this, we conducted a focused analysis of gene expression related to inflammation and autoimmunity in lung tissue.

Methods: We induced pulmonary hypertension (PH) in mice using the Sugen 5416/Hypoxia method (n=29) and then categorized them into various experimental groups: sham-induced animals without PH (control, n=8), Sugen 5416/Hypoxia-induced PH without any treatment (PH, n=5), PH-mice treated with a dual endothelin receptor antagonist (dual ERA, n=4), those receiving @ED-A⁺ Fn antibody treatment (@ED-A⁺, n=7), or treated with KSF (KSF, n=5). All animals underwent echocardiographic evaluation of right ventricular morphology and function (e.g., tricuspid annular systolic plane excursion, TAPSE) as well as right heart catheterization to assess right ventricular systolic pressure (RVP_sys). All experimental groups were subjected to RT-PCR-based gene expression analysis of inflammation and autoimmunity-related genes (a total of 84 genes) in lung tissue.

Results: In the untreated PH group, 21 genes displayed significantly (>5-fold) increased changes in expression compared to the control group. The most prominent upregulated genes included Chemokine (C-X-C-motif) receptor 1 (CXCR1; +15.5-fold), Chemokine (C-X-C-motif) ligand 1 (CXCL1; +9,2-fold), Chemokine (C-C-motif) ligand 3 (CCL3 +8-fold), Interleukin 1beta (IL1b +7.2-fold) and Tumor necrosis factor (TNF; +12.1-fold). @ED-A⁺ Fn antibody treatment, in general, was associated with less pronounced increases in gene expression levels compared to the untreated PH group. Notably, this effect was most evident for CCL3, CXCL1, Interleukin 1beta, and TNF, which exhibited a trend toward a return to normal or even decreased expression compared to the control group. Regarding those genes, similar effects were observed to a lesser extent in PH mice treated with KSF. Furthermore, the dual ERA group exhibited a milder increase in most of the genes, respecting fold changes compared to the untreated PH group, but this effect was less pronounced than that seen in @ED-A⁺ Fn antibody treated mice.

Conclusion: Correlating with augmented RVP_sys and a decrease in TAPSE, @ED-A⁺ Fn antibody treatment led to a significantly diminished upregulation of specific genes related to inflammatory response and autoimmunity in this preclinical model of PH. Thus, @ED-A⁺ Fn therapy, known for its effectiveness in treating PH in animal models, likely exerts its beneficial effects through various anti-inflammatory mechanisms, ultimately reducing pulmonary vascular remodelling.