Celastrol suppresses neovascularization in rat aortic vascular endothelial cells stimulated by inflammatory tenocytes via modulating the NLRP3 pathway

Rotator cuff injury is prevalent and burdensome, and despite surgical advances, re-tear rates remain high. Tendon-to-bone healing involves coordinated inflammation and vascular remodeling; however, excessive angiogenesis can worsen scarring and impair function. Pro-inflammatory cytokines (e.g., TNF-α, IL-1, IL-6) can drive VEGFA expression and pathological neovascularization, linking inflammation to aberrant angiogenesis during healing. The study asks whether inflammatory tenocytes promote endothelial angiogenesis and whether celastrol, a bioactive anti-inflammatory compound from Tripterygium wilfordii, can suppress this response. The central hypothesis is that celastrol promotes tendon-bone healing by attenuating inflammation-induced angiogenesis via modulation of the NLRP3 pathway.

Prior work indicates a strong association between inflammation and tendinopathy, with activation of IFN, NF-κB, and STAT pathways and elevated cytokines in diseased tendons. Pathological angiogenesis contributes to poor outcomes in tendinitis and other conditions; VEGF is a key regulator of vascular growth and permeability and is upregulated by inflammatory mediators (TNF-α, IL-6). The timing and extent of VEGF expression after tendon injury influence healing, with evidence that restricting excessive angiogenesis can reduce scarring and improve biomechanical properties at later stages. Celastrol exhibits immunosuppressive and anti-inflammatory actions across diseases (e.g., rheumatoid arthritis, osteoarthritis), acting on pathways such as NF-κB and modulating macrophage function and osteoclastogenesis. Its role in rotator cuff injury and regulation of NLRP3 inflammasome signaling in tendon contexts has been less defined, motivating the current investigation.

In vitro: Primary rat tenocytes were isolated from SD rats, expanded for 3–4 passages, and seeded at 1×10^5/mL in six-well plates. Inflammation was induced with LPS (0.5 or 1 μg/mL) for 6 or 12 h. A rat aortic vascular endothelial cell (RAOEC) line was used for angiogenesis assays. Conditioned medium (CM) from LPS-treated tenocytes was collected. Tube formation assay: 96-well plates were coated with Matrigel; RAOECs (≈10,000 cells/well) were suspended in CM or control media and incubated 6 h; capillary-like structures were imaged (Zeiss Axio Observer) and quantified with AngioTool. ELISA: VEGFA in tenocyte supernatants was quantified (Beyotime kit #PV960) using a Bio-Rad plate reader. Gene expression: Total RNA was extracted (TRIzol), reverse transcribed (TransGen kits), and qPCR performed (TransStart Top Green qPCR); primers for IL-1β, Tnf-α, Nlrp3, Vegfa, Gapdh are provided (Table 1). Protein analysis: Western blotting was performed on RIPA lysates; proteins separated by 10% SDS-PAGE and transferred to PVDF; primary antibodies: NLRP3 (Abcam ab263899), IL-1β (Abcam EPR21086), GAPDH (Abcam ab8245); secondary antibodies (Abclonal); detection by ECL (Beyotime). Immunocytochemistry: Tenocytes on poly-L-lysine-coated coverslips were fixed (4% PFA) and stained for NLRP3 (Abcam ab263899) with AlexaFluor 488 secondary; nuclei counterstained with DAPI; imaging by Zeiss LSM 880 confocal. In vivo: A rat rotator cuff tear (RCT) model was established: a 1.5 cm incision exposed supraspinatus; half of the tendon was detached from the greater tuberosity and then repaired with 4-0 sutures. Groups: Sham, RCT, RCT+Celastrol (1 mg/kg intra-articular celastrol weekly). Endpoints at 4 and 8 weeks. Biomechanics: Using MTS 858 system, preload up to 5 N, constant extension 14 mm/s; outcomes: ultimate load to failure (N) and stiffness (N/mm), analyzed via load-displacement curves. Tissue qPCR measured Vegfa and Nlrp3 mRNA in tendons. Statistics: Experiments repeated at least three times; data as mean±SD; Student’s t-test for two groups; one-way ANOVA for ≥3 groups; significance at P<0.05. Ethics: Approved by the Ethics Committee of the First Affiliated Hospital, Jinan University (202108246-25).

- LPS induced tenocyte inflammation: Time- and dose-dependent increases in Nlrp3, Tnf-α, Il-1β, and Vegfa mRNA (6 and 12 h; 0.5 or 1 μg/mL). NLRP3 and IL-1β protein levels were elevated, with the highest at 0.5 μg/mL for 12 h. VEGFA secretion in supernatants increased (ELISA), with statistical significance (P<0.05 to P<0.0001). - Conditioned medium from LPS-treated tenocytes enhanced RAOEC tube formation, increasing total tube length versus control (P<0.001). - Celastrol suppressed LPS-induced inflammatory signaling in tenocytes: reduced Nlrp3 and Il-1β mRNA and protein, and decreased NLRP3 immunofluorescence signal (P<0.05 to P<0.001). - Celastrol decreased VEGFA secretion from LPS-induced tenocytes and, correspondingly, significantly inhibited RAOEC tube formation (reduced total tube length; P<0.001), implicating a VEGFA-dependent effect. - In vivo RCT model: Weekly intra-articular celastrol (1 mg/kg) improved biomechanics at 4 and 8 weeks, increasing ultimate load to failure and stiffness relative to RCT controls (P<0.01 to P<0.0001). Tendon Vegfa and Nlrp3 mRNA elevations in RCT were reduced by celastrol at both time points (P<0.05 to P<0.0001).

The study demonstrates that inflammatory activation of tenocytes by LPS augments pro-inflammatory cytokines and VEGFA, generating a secretome that drives endothelial tube formation, thereby linking tenocyte inflammation to angiogenesis. Celastrol attenuated NLRP3 and IL-1β expression and reduced VEGFA secretion, resulting in diminished angiogenic stimulation of RAOECs. In vivo, celastrol improved mechanical properties of repaired tendons and lowered Vegfa and Nlrp3 expression, supporting the premise that moderating inflammation-induced angiogenesis via the NLRP3 axis benefits tendon-to-bone healing. These findings underscore the importance of precisely regulating neovascularization during repair—curbing excessive angiogenesis can reduce scarring and improve biomechanical recovery—and identify celastrol as a candidate modulator of this process through NLRP3 pathway regulation.

Celastrol suppresses inflammation-induced angiogenesis by downregulating NLRP3/IL-1β and VEGFA in tenocytes, reducing RAOEC tube formation in vitro, and improving biomechanical outcomes with decreased Vegfa and Nlrp3 expression in an RCT rat model. The work highlights a mechanistic link between tenocyte inflammation, VEGFA-driven neovascularization, and impaired tendon-bone healing, proposing celastrol as a potential anti-angiogenic adjunct to enhance rotator cuff repair. Future research should delineate broader molecular pathways beyond NLRP3/IL-1β (e.g., ECM remodeling, cytoskeletal dynamics), profile comprehensive cytokine and secretome changes, optimize celastrol delivery to improve solubility and bioavailability while minimizing toxicity, and validate efficacy and safety in larger animal models and clinical studies.

- The study focuses primarily on the NLRP3/IL-1β axis; downstream mechanisms tied to collagen production, ECM organization, and cytoskeletal dynamics were not interrogated. - A comprehensive panel of inflammatory mediators (e.g., IL-2, IL-6, IL-10) and additional secreted factors beyond VEGFA were not measured. - Celastrol has pharmacological limitations, including poor water solubility, low bioavailability, narrow therapeutic window, and potential adverse and immunosuppressive effects; long-term safety and optimized delivery strategies were not addressed.