MULTI-OMICS ANALYSIS REVEALS PLASMA BIOMARKER CANDIDATES FOR CALCIFIC UREMIC ARTERIOLOPATHY PATIENTS TREATED WITH HUMAN AMNION-DERIVED MESENCHYMAL STEM CELLS

Introduction:

Calciphylaxis, also known as calcific uremic arteriolopathy (CUA) when it occurs in chronic kidney disease (CKD) patients, is a progressive ischemic cutaneous orphan disease (ORPHA:280062). There is no proven therapy with a one-year mortality rate of up to 80%. Identifying non-invasive biomarkers is essential for optimizing novel treatments.

Methods:

We rescued CUA patients with human amnion-derived mesenchymal stem cells (hAMSCs). In a discovery cohort with uremic patients (n=10) and CUA patients (n=3), we conducted an in-depth plasma proteomic analysis to compare the differentially expressed proteins (DEPs). Dynamic changes in the plasma proteome of a CUA patient treated with hAMSCs for 15 months were monitored. Single-cell transcriptome sequencing of peripheral blood mononuclear cells (PBMCs) was conducted. Plasma biomarker candidates were measured by ELISA in both the discovery and verification cohort. Skin tissue was analyzed by immunohistochemistry (IHC) staining (Fig. 1).

Fig. 1. Study design.

Results:

Patient 1 (Fig. 2A-2F) and patient 3 (Fig. 2J-2L) achieved skin recovery after hAMSC therapy. Patient 2 terminated hAMSC treatment due to the COVID-19 pandemic (Fig. 2G-2I).

Fig. 2. Skin lesions of CUA patients treated with hAMSCs in the discovery cohort. 

As the core protein in plasma DEP network, Thrombospondin 1 (THBS1 or Tsp-1) was upregulated in the CUA group (Fig. 3).

Fig. 3. Static proteomic analysis showed a crucial role of THBS1 in CUA plasma.

Plasma dynamic proteomic analysis revealed that THBS1 levels were significantly decreased after hAMSC therapy for 3 days. Latent transforming growth factor-beta (TGF-β)-binding protein 1 (LTBP1) exhibited the same trend (Fig. 4A-4C). We clustered all plasma proteins using Mfuzz. Cluster 4 shared the same changes as THBS1 and LTBP1. Gene ontology (GO) analysis revealed a high association with “wound healing”, “blood coagulation” and “platelet alpha granule” (Fig. 4D-4E). THBS1 and TGF-β1 emerged as hub proteins in the network (Fig. 4F) .

Fig. 4. Plasma core DEPs and enriched pathways in the CUA patient treated with hAMSCs.

THBS1 and TGFB1 were mainly produced by megakaryocytes of the CUA patient, rapidly downregulated after hAMSC treatment (Fig. 5A-5D). Compared to the uremic group, the plasma levels of THBS1/TGF-β1, measured by ELISA, were increased 11.75- and 10.75-fold, respectively, in the CUA group (Fig. 5E). After hAMSC therapy, while plasma THBS1/TGF-β1 levels continued to be low in the CUA patient, there was a gradual rise as the stem cell treatment frequency decreased (Fig. 5F).

Fig. 5. The source of THBS1 and TGFB1 in the CUA patient revealed by single-cell transcriptome sequencing and their plasma levels in the discovery cohort.

In the validation cohort, the plasma levels of THBS1/TGF-β1 in the CUA group (n=7) were higher than in the uremia group (n=20), with area under the curve (AUC) values of 1 and 0.986, respectively. These levels can be inhibited after three days of hAMSC treatment (Fig. 6A-6B). Compared with the healthy control, THBS1 and its receptor, CD47, were increased in the CUA skin (Fig. 6C-6L).

Fig. 6. Biological experiments of the independent validation cohort.

Conclusions:

Plasma THBS1 and TGF-β1 are biomarker candidates for CUA patients. Personalized hAMSC strategies involve intravenous delivery and are based on biomarker levels during the regular follow-up. 

I have no potential conflict of interest to disclose.

I did not use generative AI and AI-assisted technologies in the writing process.