Biology subjects

Dietmar, H. F.

Publications and source records attributed to Dietmar, H. F..

3 recordsLinked to original sources

bioRxiv2025

Dichotomous SMAD2/3 regulation and selective anti-hypertrophic activity of heparin during in vitro chondrogenesis of mesenchymal stromal cells

BackgroundEndochondral instead of chondral differentiation hinders mesenchymal stromal cell (MSC) application for clinical cartilage regeneration. We previously showed that heparin-polyethylene glycol (PEG) hydrogels loaded with transforming growth factor beta (TGF-{beta}) instructed stable chondral MSC development in vivo. We here assessed this approach in vitro, utilizing heparin-PEG hydrogels or soluble heparin supplementation of chondrogenic medium. MethodsHuman MSCs were cultured in heparin-PEG hydrogels (22.4 mg/mL crosslinked heparin, 120ng TGF-{beta}1) or as hydrogel-free pellet cultures treated with soluble heparin (0, 10, 100, 700 g/mL) in TGF-{beta}1-containing (10 ng/mL) chondrogenic medium. Chondral and endochondral signaling (1-3 h, 4 weeks) and cartilage matrix formation (4 weeks) were analyzed using Western blot, histology, qPCR, ELISA, and enzyme activity. ResultsUnlike in vivo, human MSCs differentiated in heparin-PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in vitro. Interestingly, treatment with soluble heparin (10-700 {micro}g/mL) revealed reduced TGF-{beta}-small mother against decapentaplegic (SMAD)3 but not SMAD2 activation at unaffected type II collagen and proteoglycan/DNA levels. We propose that the stimulation of the insulin-AKT pathway by heparin aided in maintaining SMAD2 activation which apparently plays a more prominent role than SMAD3 for MSC chondrogenesis. Heparin treatment inhibited the pro-hypertrophic WNT/{beta}-catenin pathway in vitro but insufficiently silenced TGF-{beta}-SMAD1/5/9 activation and unfortunately reduced anti-hypertrophic prostaglandin E2 (PGE2) levels. Ultimately, treatment with 10 {micro}g/mL heparin reduced the upregulation of several hypertrophy markers (MEF2C, IHH, IBSP mRNAs, alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous anti-hypertrophic interventions (parathyroid-hormone related protein (PTHrP)-pulses, wingless-int (WNT)-inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation. ConclusionsTaken together, heparin-TGF-{beta} showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic power and context-dependent lineage-instructive properties: permitting endochondral differentiation in vitro but chondral development in vivo. Thus, environmental contributions are mandatory to allow heparin-PEG-guided chondral versus endochondral lineage commitment of MSCs in vivo, potentially involving SMAD1/5/9 suppressors and PGE2 sources. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/673657v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@140313aorg.highwire.dtl.DTLVardef@160049dorg.highwire.dtl.DTLVardef@cee22corg.highwire.dtl.DTLVardef@62e961_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2024

The neuropeptide aCGRP impairs the chondrocyte response to mechanical load

ObjectiveNovel targets for osteoarthritis therapy are urgently needed, and sensory nerve fibres and their neuropeptides are increasingly recognised for their contribution to structural aspects of joint pathology. The nociceptive sensory neuropeptide alpha calcitonin gene-related peptide (CGRP) was previously detected in synovial fluid and serum of osteoarthritis patients and was also described as trophic factor for chondrocytes, affecting ECM organisation and biomechanical properties. Here, we investigated the potential of CGRP to alter the chondrocyte mechanoresponse, and thus to affect the resilience of cartilage towards mechanical loading. MethodsTissue-engineered neocartilage based on human articular chondrocytes was treated with 1{micro}M CGRP for 24 hours and subjected to an anabolic loading protocol (intermittent dynamic compression, 1Hz, 25%) for the last 3 hours before analysing its molecular mechano-response. ResultsMechanotransduction was largely unaltered by CGRP as demonstrated by ERK activation and stimulation of mechano-regulated gene expression, yet load-stimulated glycosaminoglycan synthesis was disturbed by CGRP. Presence of CGRP did not affect stimulation of WNT5A expression by loading, but decreased DKK3 expression under loading. Importantly, WNT inhibition prevented the negative effect of CGRP on load-stimulated glycosaminoglycan synthesis. ConclusionIdentifying WNT5A as a novel mechano-response gene, we reveal that CGRP can block the load-stimulated proteoglycan production of human chondrocytes in the presence of WNT pathway activity. Thus, our data propose a novel, negative role for the pain-mediator CGRP in the cartilage loading response, compromising its resilience to loading. Overall, our study implicates CGRP as a potential target for osteoarthritis treatment, but also in patient stratification.

cell biology↗

bioRxiv2024

Load activated FGFR and beta1 integrins target distinct chondrocyte mechano-response genes

In response to mechanical stimuli, chondrocytes adapt their transcriptional activity, thereby shaping the cellular mechano-response; however, it remains unclear whether the activation of cell surface receptors during mechanical loading converge in the activation of the same mechano-response genes, or whether pathway-specific genes can be defined. We aimed to determine whether load-activated FGF/FGFR signalling and {beta}1 integrin jointly activate ERK and control the same or distinct subsets of mechano-regulated genes. To this end, tissue-engineered neocartilage was generated from murine costal chondrocytes or human articular chondrocytes and subjected to dynamic unconfined compression with or without FGFR inhibition. To assess the role of {beta}1 integrins, neocartilage was generated from embryonic {beta}1 integrin-deficient or wild type costal chondrocytes. Load-activated FGFR signalling drove ERK activation in murine chondrocytes, and partially also in human chondrocytes, and mechano-response genes could be classified according to their regulation: Fosl1, Itga5, Ngf and Timp1 were regulated by load-activated FGFR depending on the developmental stage, whereas {beta}1 integrins controlled Inhba expression. In human chondrocytes, load-activated FGFR controlled expression of BMP2, PTGS2 and DUSP5, but not FOSB. We show here that the chondrocyte loading response is coordinated by concurrent activation of multiple receptors, and identified for the first time distinct target genes of activated receptors. These insights open up the opportunity to pharmacologically shape the mechano-response of chondrocytes in future studies with promising implications for the management of osteoarthritis and the development of novel therapeutic strategies.

cell biology↗