Multiomic study of skin, peripheral blood, and serum: is serum proteome a reflection of disease process at the end-organ level in systemic sclerosis?

BACKGROUND: Serum proteins can be readily assessed during routine clinical care. However, it is unclear to what extent serum proteins reflect the molecular dysregulations of peripheral blood cells (PBCs) or affected end-organs in systemic sclerosis (SSc). We conducted a multiomic comparative analysis of SSc serum profile, PBC, and skin gene expression in concurrently collected samples. METHODS: Global gene expression profiling was carried out in skin and PBC samples obtained from 49 SSc patients enrolled in the GENISOS observational cohort and 25 unaffected controls. Levels of 911 proteins were determined by Olink Proximity Extension Assay in concurrently collected serum samples. RESULTS: Both SSc PBC and skin transcriptomes showed a prominent type I interferon signature. The examination of SSc serum profile revealed an upregulation of proteins involved in pro-fibrotic homing and extravasation, as well as extracellular matrix components/modulators. Notably, several soluble receptor proteins such as EGFR, ERBB2, ERBB3, VEGFR2, TGFBR3, and PDGF-Rα were downregulated. Thirty-nine proteins correlated with severity of SSc skin disease. The differential expression of serum protein in SSc vs. control comparison significantly correlated with the differential expression of corresponding transcripts in skin but not in PBCs. Moreover, the differentially expressed serum proteins were significantly more connected to the Well-Associated-Proteins in the skin than PBC gene expression dataset. The assessment of the concordance of between-sample similarities revealed that the molecular profile of serum proteins and skin gene expression data were significantly concordant in patients with SSc but not in healthy controls. CONCLUSIONS: SSc serum protein profile shows an upregulation of profibrotic cytokines and a downregulation of soluble EGF and other key receptors. Our multilevel comparative analysis indicates that the serum protein profile in SSc correlates more closely with molecular dysregulations of skin than PBCs and might serve as a reflection of disease severity at the end-organ level.

Neutrophil Elastase-Generated Fragment of Vascular Endothelial Growth Factor-A Stimulates Macrophage and Endothelial Progenitor Cell Migration.

Elastase released from neutrophils as part of the innate immune system has been implicated in chronic diseases such as emphysema and cardiovascular disease. We have previously shown that neutrophil elastase targets vascular endothelial growth factor-A (VEGF) for partial degradation to generate a fragment of VEGF (VEGFf) that has distinct activities. Namely, VEGFf binds to VEGF receptor 1 but not to VEGF receptor 2 and shows altered signaling compared to intact VEGF. In the present study we investigated the chemotactic function of VEGF and VEGFf released from cells by neutrophil elastase. We found that endothelial cells migrated in response to intact VEGF but not VEGFf whereas RAW 264.7 macrophages/monocytes and embryonic endothelial progenitor cells were stimulated to migrate by either VEGF or VEGFf. To investigate the role of elastase-mediated release of VEGF from cells/extracellular matrices, a co-culture system was established. High or low VEGF producing cells were co-cultured with macrophages, endothelial or endothelial progenitor cells and treated with neutrophil elastase. Elastase treatment stimulated macrophage and endothelial progenitor cell migration with the response being greater with the high VEGF expressing cells. However, elastase treatment led to decreased endothelial cell migration due to VEGF cleavage to VEGF fragment. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment.

Complex receptor-ligand dynamics control the response of the VEGF system to protease injury.

BACKGROUND: Vascular homeostasis and response to injury are dependent on the coordinated activity of growth factors such as vascular endothelial growth factor-A (VEGF). VEGF signaling is mediated by VEGF receptors 1 (VEGFR1) and 2 (VEGFR2). VEGF also binds to extracellular matrix (ECM) and neuropilin (NP), a cell surface glycoprotein that enhances VEGF binding to VEGFR2 while inhibiting VEGF-VEGFR1 interactions. Proteases such as neutrophil elastase release VEGF bound to ECM; however, this results in proteolytic processing of VEGF to a smaller species termed VEGF fragment (VEGFf). We hypothesized that the generation and presence of VEGFf would have significant effects on the binding distribution of VEGF. RESULTS: We show that VEGFf, unlike VEGF, does not bind ECM, fibronectin, or NP-1. Using computational simulations, we find that excess VEGFf can lead to increased binding of VEGF to VEGFR2 through VEGFf binding to VEGFR1 and subsequent liberation of NP-1. We show experimentally that VEGF-induced migration has a biphasic response to conversion of VEGF to VEGFf. Simulations suggest that a simple change in VEGFR1 or VEGFR2 complexes are unlikely to be responsible and that a more complex integration of signals is more likely involved. CONCLUSIONS: These findings suggest that proteolytic damage at sites of tissue injury and inflammation has the potential to modulate the VEGF system through a complex process and highlight the need for quantitative analysis to reveal mechanisms of growth factor control.

Engineered bivalent ligands to bias ErbB receptor-mediated signaling and phenotypes.

The ErbB receptor family is dysregulated in many cancers, and its therapeutic manipulation by targeted antibodies and kinase inhibitors has resulted in effective chemotherapies. However, many malignancies remain refractory to current interventions. We describe a new approach that directs ErbB receptor interactions, resulting in biased signaling and phenotypes. Due to known receptor-ligand affinities and the necessity of ErbB receptors to dimerize to signal, bivalent ligands, formed by the synthetic linkage of two neuregulin-1ß (NRG) moieties, two epidermal growth factor (EGF) moieties, or an EGF and a NRG moiety, can potentially drive homotypic receptor interactions and diminish formation of HER2-containing heterodimers, which are implicated in many malignancies and are a prevalent outcome of stimulation by native, monovalent EGF, or NRG. We demonstrate the therapeutic potential of this approach by showing that bivalent NRG (NN) can bias signaling in HER3-expressing cancer cells, resulting in some cases in decreased migration, inhibited proliferation, and increased apoptosis, whereas native NRG stimulation increased the malignant potential of the same cells. Hence, this new approach may have therapeutic relevance in ovarian, breast, lung, and other cancers in which HER3 has been implicated.

Neutrophil elastase cleaves VEGF to generate a VEGF fragment with altered activity.

Excessive neutrophil elastase (NE) activity and altered vascular endothelial growth factor (VEGF) signaling have independently been implicated in the development and progression of pulmonary emphysema. In the present study, we investigated the potential link between NE and VEGF. We noted that VEGF(165) is a substrate for NE. Digestion of purified VEGF(165) with NE generated a partially degraded disulfide-linked fragment of VEGF. Mass spectrometric analysis revealed that NE likely cleaves VEGF(165) at both the NH(2) and COOH termini to produce VEGF fragment chains approximately 5 kDa reduced in size. NE treatment of VEGF-laden endothelial cell cultures and smooth muscle cells endogenously expressing VEGF generated VEGF fragments similar to those observed with purified VEGF(165). NE-generated VEGF fragment showed significantly reduced binding to VEGF receptor 2 and heparin yet retained the ability to bind to VEGF receptor 1. Interestingly, VEGF fragment showed altered signaling in pulmonary artery endothelial cells compared with intact VEGF(165). Specifically, treatment with VEGF fragment did not activate extracellular-regulated kinases 1 and 2 (ERK1/2), yet resulted in enhanced activation of protein kinase B (Akt). Treatment of monocyte/macrophage RAW 264.7 cells with VEGF fragment, on the other hand, led to both Akt and ERK1/2 activation, increased VEGFR1 expression, and stimulated chemotaxis. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment and activation via VEGF receptor 1.