127 - EVALUATION OF MARKERS OF ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH KAWASAKI DISEASE ON FOLLOW-UP USING FLOWCYTOMETRY, TRANSCRIPTOMICS AND PROTEOMICS- THE FIRST SUCH STUDY FROM THE INDIAN SUBCONTINEN

Background

Endothelial dysfunction (ED) is a known complication of KD. However its etiopathogenesis remains obscure. Endothelium maintains vascular homeostasis but pathological conditions result in detachment of endothelial cells (ECs) from vessel wall into circulation. ED has been assessed using ultrasonography and other clinical parameters. However, there is paucity of literature on laboratory-based assessment of ED in KD patients on follow-up.  In this study, we evaluate ED by exploring circulating cells and their progenitors, angiogenesis factors, cytokines, cell-adhesion molecules and adipocytokine markers.

Methods

This is a single centre, prospective study conducted from July 2017 to July 2022. We enrolled 65 patients of KD as per AHA 2017, and 20 age matched healthy controls (HC). Patients were categorised as follows:  Group 1: (n=19; >6 months -1.5 years on follow-up); Group 2: (n=24; >1.5 - 3 years on follow-up); and Group 3: (n=22; >3 - 4.5 years on follow-up). Twenty-nine patients had CAAs [transient (n=13), persistent (n=16)] and 36 patients had no CAAs. Characterization of EPCs (CD34⁺/CD309⁺/CD133⁺) and CECs (CD45^dim/CD146⁺/CD31⁺/CD133^-) was done using flowcytometry. (Figure 1a, 1d). Cell sorting was carried out for EPCs and CECs (n=6; 3KD, 3HC) for bulk RNA-seq. Differentially expressed genes (DEGs) specific to ED (VEGFA, RETN, PTX3, LEP, ENG, SPP1, CXCL8, PECAM1) were selected and validated on whole blood (n=65KD, 20HC) using SYBR green chemistry. Protein validation of analytes was done using Luminex based platform (Multiplexing) and by ELISA.

Results

Statistical analysis showed higher number and percentage of EPCs (Gp1: p-0.02, Gp2: p< 0.0001) and CECs (Gp1: p< 0.0001, Gp2: p< 0.0001, Gp3: p-0.001) in KD patients when compared to HC (Figure 1c, 1f), and in patients with CAAs when compared to those without CAAs (Figure1b, 1e). DEGs from sorted cells revealed promising genes. Quantitative gene expression of CXCL8, PTX3 and VEGFA was elevated in KD patients (Figure 2). Protein validation showed increased levels of angiopoietin, pecam-1, pentraxin-3, endoglin, CXCL8, resistin and osteopontin in KD patients when compared to HC (Figure 3) and in patients with persistent CAAs as compared to patients with transient CAAs.

Conclusion
Increased number of EPCs and CECs, and elevated levels of the assayed analytes, suggest persistence of ED even after the acute stage of KD. These parameters can serve as disease biomarkers during long-term follow-up of patients with KD. These may be taken as pointers towards emergence of CAAs in patients with KD who did not have any overt abnormality during acute stage.