Oxidative stress-induced damage of the retinal pigment epithelium (RPE) and chronic inflammation have been suggested as major contributors to a range of retinal diseases. Here, we examined the effects of oxidative stress on endogenous complement components and proinflammatory and angiogenic responses in RPE cells. ARPE-19 cells exposed for 1–48 h to H₂O₂ had reduced cell–cell contact and increased markers for epithelial–mesenchymal transition but showed insignificant cell death. Stressed ARPE-19 cells increased the expression of complement receptors CR3 (subunit CD11b) and C5aR1. CD11b was colocalized with cell-derived complement protein C3, which was present in its activated form in ARPE-19 cells. C3, as well as its regulators complement factor H (CFH) and properdin, accumulated in the ARPE-19 cells after oxidative stress independently of external complement sources. This cell-associated complement accumulation was accompanied by increased nlrp3 and foxp3 expression and the subsequently enhanced secretion of proinflammatory and proangiogenic factors. The complement-associated ARPE-19 reaction to oxidative stress, which was independent of exogenous complement sources, was further augmented by the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib. Our results indicate that ARPE-19 cell-derived complement proteins and receptors are involved in ARPE-19 cell homeostasis following oxidative stress and should be considered as targets for treatment development for retinal degeneration.

The retinal pigment epithelium (RPE) maintains visual function and preserves structural integrity of the retina. Chronic dysfunction of the RPE is associated with retinal degeneration, including age-related macular degeneration (AMD). The AMD pathogenesis includes both increased oxidative stress and complement dysregulation. Physiological sources of oxidative stress in the retina are well known, while complement sources and regulation are still under debate. Using human primary RPE (hpRPE) cells, we have established a model to investigate complement component expression on transcript and protein level in AMD-risk and non-risk hpRPE cells. We evaluated the effect of properdin, a complement stabilizer, on the hpRPE cell-dependent complement profile exposed to oxidative stress. hpRPE cells expressed complement components, receptors and regulators. Complement proteins were also stored and secreted by hpRPE cells. We associated AMD-risk single nucleotide polymorphisms with an increased secretion of complement factors D (CFD) and I (CFI). Furthermore, we detected hpRPE cell-associated complement activation products (C3a, C5a) independent of any extracellularly added complement system. Exogenous properdin increased the mRNA expression of CFI and CFD, but decreased levels of complement components (C1Q, C3), receptors (C3AR, C5AR1, CD11B) and inflammation-associated transcripts (NLRP3, IL1B) in hpRPE cells exposed to oxidative stress. This properdin effect was time-dependently counter regulated. In conclusion, our data unveiled a local, genotype-associated complement component production in hpRPE cells, regulated by exogenous properdin. The local complement production and activation via blood-independent mechanisms can be a new therapeutic target for AMD.

Bone marrow-derived mesenchymal stromal cells (BMSCs) respond to a variety of tumor cell-derived signals, such as inflammatory cytokines and growth factors. As a result, the inflammatory tumor microenvironment may lead to the recruitment of BMSCs. Whether BMSCs in the tumor environment are more likely to promote tumor growth or tumor suppression is still controversial. In our experiments, direct 3D co-culture of BMSCs with tumor cells from the head and neck region (HNSCC) results in strong expression and secretion of MMP-9. The observed MMP-9 secretion mainly originates from BMSCs, leading to increased invasiveness. In addition to our in vitro data, we show in vivo data based on the chorioallantoic membrane (CAM) model. Our results demonstrate that MMP-9 induces hemorrhage and increased perfusion in BMSC/HNSCC co-culture. While we had previously outlined that MMP-9 expression and secretion originate from BMSCs, our data showed a strong downregulation of MMP-9 promoter activity in HNSCC cells upon direct contact with BMSCs using the luciferase activity assay. Interestingly, the 2D and 3D models of direct co-culture suggest different drivers for the downregulation of MMP-9 promoter activity. Whereas the 3D model depicts a BMSC-dependent downregulation, the 2D model shows cell density-dependent downregulation. In summary, our data suggest that the direct interaction of HNSCC cells and BMSCs promotes tumor progression by significantly facilitating angiogenesis via MMP-9 expression. On the other hand, data from 3D and 2D co-culture models indicate opposing regulation of the MMP-9 promoter in tumor cells once stromal cells are involved.

Introduction: Orthobiologics, such as autologous nanofat, are emerging as a potential treatment option for osteoarthritis (OA), a common degenerative joint causing pain and disability in the elderly. Nanofat, a minimally processed human fat graft rich in stromal vascular fraction (SVF) secretory factors, has shown promise in relieving pain. This study aimed to elucidate the molecular mechanisms underlying nanofat treatment of OA-affected cells and compare two filtration systems used for nanofat preparation.Methods: Chondrocytes and synoviocytes were isolated from articular cartilage and synovium of 22 OA-patients. Lipoaspirates from 13 OA-patients were emulsified using the Adinizer^® or Lipocube^™ Nano filter systems to generate nanofat. The fluid phase of SVF from both filtered and unfiltered lipoaspirates was applied to OA-affected cells. Luminex multiplex ELISA were performed with lipoaspirates and cell supernatants alongside functional assays evaluating cell migration, proliferation, metabolic activity, and senescence.Results: A total of 62 cytokines, chemokines, growth factors, neuropeptides, matrix-degrading enzymes, and complement components were identified in lipoaspirates. Among these, significant concentration differences were observed for TIMP-2, TGF-ß₃, and complement component C3 between the filtered and unfiltered samples. Nanofat enhanced chondrocyte proliferation and migration, as well as synoviocyte migration and metabolic activity, while reducing chondrocyte metabolic activity. Pain-related factors like β-NGF, MCP-1, Substance P, VEGF, and αCGRP were reduced, while anti-inflammatory TGF-β₁₊₃ increased and pro-inflammatory cytokines (IL-5, IL-7, IL-15, and IFN-γ) decreased. Nanofat also elevated secretion of complement components and TIMPs in both cell types. Notably, our results revealed no significant differences in cellular effects between sSVF filtered using the Adinizer^® and Lipocube^™ Nano systems, as well as compared to unfiltered sSVF.Discussion: Here, we provide first insights into how autologous nanofat therapy may ameliorate OA by enhancing chondrocyte proliferation and synoviocyte migration while modulating inflammatory and pain-related factors. However, further research is needed to determine its effects on cartilage regeneration.

Retinal degeneration is associated with complement system activation, but retinal sources of complement are unknown. Here, we describe the human and murine complement transcriptomes of Müller cells, microglia/macrophages, vascular cells, neurons and retinal pigment epithelium (RPE) in health and disease. All cell populations expressed c1s, c3, cfb, cfp, cfh and cfi . Murine Müller cells contributed the highest amount of complement activators ( c1s, c3, cfb ). RPE mainly expressed cfh, while cfi and cfp transcripts were most abundant in neurons. The main complement negative regulator in the human retina was cfi , while cfh dominated in the murine retina. Importantly, the expression of c1s, cfb, cfp, cfi increased and that of cfh decreased with aging. Impaired photoreceptor recycling led to an enhanced c3 expression in RPE and to a reduced cfi expression in microglia/macrophages. Expression of complement components was massively upregulated after transient retinal ischemia in murine microglia, Müller cells and RPE. The individual signature of complement expression in distinct murine and human retinal cell types indicates a local, well-orchestrated regulation of the complement system in both species.