Miriam Boersema

Signaling in Fibrosis: TGF-β, WNT, and YAP/TAZ Converge

Chronic organ injury leads to fibrosis and eventually organ failure. Fibrosis is characterized by excessive synthesis, remodeling, and contraction of extracellular matrix produced by myofibroblasts. Myofibroblasts are the key cells in the pathophysiology of fibrotic disorders and their differentiation can be triggered by multiple stimuli. To develop anti-fibrotic therapies, it is of paramount importance to understand the molecular basis of the signaling pathways contributing to the activation and maintenance of myofibroblasts. Several signal transduction pathways, such as transforming growth factor (TGF)-β, Wingless/Int (WNT), and more recently yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling, have been linked to the pathophysiology of fibrosis. Activation of the TGF-β1-induced SMAD complex results in the upregulation of genes important for myofibroblast function. Similarly, WNT-stabilized β-catenin translocates to the nucleus and initiates transcription of its target genes. YAP and TAZ are two transcriptional co-activators from the Hippo signaling pathway that also rely on nuclear translocation for their functioning. These three signal transduction pathways have little molecular similarity but do share one principle: the cytosolic/nuclear regulation of its transcriptional activators. Past research on these pathways often focused on the isolated cascades without taking other signaling pathways into account. Recent developments show that parts of these pathways converge into an intricate network that governs the activation and maintenance of the myofibroblast phenotype. In this review, we discuss the current understanding on the signal integration between the TGF-β, WNT, and YAP/TAZ pathways in the development of organ fibrosis. Taking a network-wide view on signal transduction will provide a better understanding on the complex and versatile processes that underlie the pathophysiology of fibrotic disorders.

Interleukin-1β attenuates myofibroblast formation and extracellular matrix production in dermal and lung fibroblasts exposed to transforming growth factor-β1.

One of the most potent pro-fibrotic cytokines is transforming growth factor (TGFβ). TGFβ is involved in the activation of fibroblasts into myofibroblasts, resulting in the hallmark of fibrosis: the pathological accumulation of collagen. Interleukin-1β (IL1β) can influence the severity of fibrosis, however much less is known about the direct effects on fibroblasts. Using lung and dermal fibroblasts, we have investigated the effects of IL1β, TGFβ1, and IL1β in combination with TGFβ1 on myofibroblast formation, collagen synthesis and collagen modification (including prolyl hydroxylase, lysyl hydroxylase and lysyl oxidase), and matrix metalloproteinases (MMPs). We found that IL1β alone has no obvious pro-fibrotic effect on fibroblasts. However, IL1β is able to inhibit the TGFβ1-induced myofibroblast formation as well as collagen synthesis. Glioma-associated oncogene homolog 1 (GLI1), the Hedgehog transcription factor that is involved in the transformation of fibroblasts into myofibroblasts is upregulated by TGFβ1. The addition of IL1β reduced the expression of GLI1 and thereby also indirectly inhibits myofibroblast formation. Other potentially anti-fibrotic effects of IL1β that were observed are the increased levels of MMP1, −2, −9 and −14 produced by fibroblasts exposed to TGFβ1/IL1β in comparison with fibroblasts exposed to TGFβ1 alone. In addition, IL1β decreased the TGFβ1-induced upregulation of lysyl oxidase, an enzyme involved in collagen cross-linking. Furthermore, we found that lung and dermal fibroblasts do not always behave identically towards IL1β. Suppression of COL1A1 by IL1β in the presence of TGFβ1 is more pronounced in lung fibroblasts compared to dermal fibroblasts, whereas a higher upregulation of MMP1 is seen in dermal fibroblasts. The role of IL1β in fibrosis should be reconsidered, and the differences in phenotypical properties of fibroblasts derived from different organs should be taken into account in future anti-fibrotic treatment regimes.

Donor and recipient origin of mesenchymal and endothelial cells in chronic renal allograft remodeling.

Chronic transplant dysfunction (CTD) is the leading cause for limited kidney graft survival. Renal CTD is characterized by interstitial and vascular remodeling leading to interstitial fibrosis, tubular atrophy and transplant vasculopathy (TV). The origin of cells and pathogenesis of interstitial and vascular remodeling are still unknown. To study graft‐versus‐recipient origin of interstitial myofibroblasts, vascular smooth muscle cells (SMCs) and endothelial cells (ECs), we here describe a new rat model for renal CTD using Dark Agouti kidney donors and R26 human placental alkaline phosphatase transgenic Fischer344 recipients. This model showed the development of CTD within 12 weeks after transplantation. In interstitial remodeling, both graft‐ and recipient‐derived cells contributed to a similar extent to the accumulation of myofibroblasts. In arteries with TV, we observed graft origin of neointimal SMCs and ECs, whereas in peritubular and glomerular capillaries, we detected recipient EC chimerism. These data indicate that, within the interstitial and vascular compartments of the transplanted kidney, myofibroblasts, SMCs and ECs involved in chronic remodeling are derived from different sources and suggest distinct pathogenetic mechanisms within the renal compartments.

Donor and Recipient Contribution to Transplant Vasculopathy in Chronic Renal Transplant Dysfunction

Background. Chronic transplant dysfunction is the leading cause of long-term renal allograft loss. One of the histologic hallmarks of chronic transplant dysfunction is transplant vasculopathy characterized by accumulation of smooth muscle cells (SMCs) in the arterial subendothelial space, leading to ischemic graft failure. Currently, no therapy is available for transplant vasculopathy, and knowledge of the origin (donor vs. recipient) of neointimal cells may contribute to develop adequate strategies. Methods. Origin of neointimal SMCs, endothelial, and tubular cells was determined in four nephrectomy samples from male recipients transplanted with a female kidney. Recipient-derived cells were detected using X- and Y-chromosome-specific fluorescent in situ hybridization combined with immunofluorescent staining. Specificity and sensitivity of fluorescent in situ hybridization were determined with corresponding controls. Results. No Y-chromosome-positive cells were detected in the female to female graft, whereas approximately 31% of nucleated cells in male to male grafts had a detectable Y-chromosome. In female to male grafts, a recipient-derived population of neointimal &agr;-smooth muscle actin-positive SMCs were detected (6%, range 3%–11%). Percentages of recipient-derived arterial endothelial cells, glomerular endothelial cells, and tubular epithelial cells were 14% (range 4%–32%), 19% (range 7%–31%) and 3% (range 2%–5%), respectively. Conclusions. Both donor- and recipient-derived cells contribute to vascular remodeling in clinical renal transplantation. The presence of &agr;-smooth muscle actin in donor- and recipient-derived cells supports a constructive role for these cells in neointimal formation. However, the predominance of donor-derived cells in the neointima points to these cells as the likely therapeutic target.

Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

Significance Collagens are structural extracellular matrix proteins that provide mechanical support to tissues. To gain stability, collagens can form pyridinoline cross-links via enzymatically formed intermediates initiated by lysyl hydroxylase (LH) 2. Individuals with mutations in the gene encoding LH2 share highly overlapping traits with individuals with mutations in the gene encoding the immunophilin FKBP65 that shows no LH activity. We found that FKBP65 is necessary for the dimerization of LH2, which is required for activity of LH2. Collagen cross-linking plays important roles in bone diseases as well as in such pathologies as cancer and fibrosis. Our study has elucidated a mechanism of how to interfere in a specific type of collagen cross-linking and can help advance the design of new treatments toward these pathologies. Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer.

Local Medial Microenvironment Directs Phenotypic Modulation of Smooth Muscle Cells After Experimental Renal Transplantation

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft‐derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation‐induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low‐density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf β1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf β1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF‐BB‐stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α‐Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF‐BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Renal heparan sulfate proteoglycans modulate fibroblast growth factor 2 signaling in experimental chronic transplant dysfunction.

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Evaluating the antifibrotic potency of galunisertib in a human ex vivo model of liver fibrosis

Liver fibrosis is a major cause of liver‐related mortality and, so far, no effective antifibrotic drug is available. Galunisertib, a TGF‐β receptor type I kinase inhibitor, is a potential candidate for the treatment of liver fibrosis. Here, we evaluated the potency of galunisertib in a human ex vivo model of liver fibrosis.

Incomplete Restoration of Angiotensin II - Induced Renal Extracellular Matrix Deposition and Inflammation Despite Complete Functional Recovery in Rats

Some diseases associated with a temporary deterioration in kidney function and/or development of proteinuria show an apparently complete functional remission once the initiating trigger is removed. While it was earlier thought that a transient impairment of kidney function is harmless, accumulating evidence now suggests that these patients are more prone to developing renal failure later in life. We therefore sought to investigate to what extent renal functional changes, inflammation and collagen deposition are reversible after cessation of disease induction, potentially explaining residual sensitivity to damage. Using a rat model of Angiotensin II (Ang II)-induced hypertensive renal disease we show the development of severe hypertension (212 ± 10.43 vs. 146 ± 1.4 mmHg, p<0.001) and proteinuria (51.4 ± 6.3 vs. 14.7 ± 2.0 mg/24h, p<0.01) with declined creatinine clearance (2.0 ± 0.5 vs. 4.9 ± 0.6 mL/min, p<0.001) to occur after 3 weeks of Ang II infusion. At the structural level, Ang II infusion resulted in interstitial inflammation (18.8 ± 4.8 vs. 3.6 ± 0.5 number of macrophages, p<0.001), renal interstitial collagen deposition and lymphangiogenesis (4.1 ± 0.4 vs. 2.2 ± 0.4 number of lymph vessels, p<0.01). Eight weeks after cessation of Ang II, all clinical parameters, pre-fibrotic changes such as myofibroblast transformation and increase in lymph vessel number (lymphangiogenesis) returned to control values. However, glomerular desmin expression, glomerular and periglomerular macrophages and interstitial collagens remained elevated. These dormant abnormalities indicate that after transient renal function decline, inflammation and collagen deposition may persist despite normalization of the initiating pathophysiological stimulus perhaps rendering the kidney more vulnerable to further damage.

CD16(+) monocytes with smooth muscle cell characteristics are reduced in human renal chronic transplant dysfunction

In chronic transplant dysfunction (CTD), persistent (allo)immune-mediated inflammation eventually leads to tissue remodeling including neointima formation in intragraft arteries. We previously showed that recipient-derived neointimal α-SMA(+) smooth muscle-like cells are present in human renal allografts with CTD. Human PBMC contain myeloid cells capable of differentiating into α-SMA(+) cells in vitro; the phenotype of the ancestral subset is as yet unknown. This study aimed to investigate whether monocyte subsets contain cells with smooth muscle-like cell differentiation capacity and whether CTD in renal transplant recipients is associated with a shift in these monocyte subsets. To accomplish this goal, monocyte subsets from healthy controls were sorted based on CD14 and CD16 expression to investigate gene expression levels of mesenchymal markers α-SMA and SM22α. CD14(+)/CD16(++) monocytes displayed increased α-SMA and SM22α mRNA expression compared with CD14(++)/CD16(-) monocytes, suggesting increased differentiation potential toward smooth muscle-like cells. Flow cytometry revealed that in non-CTD transplant recipients the percentage of CD14(+)/CD16(++) monocytes was reduced, with an even further reduction in patients with CTD. To determine a potential correlation between CD14(+)/CD16(++) monocytes and α-SMA(+) cell outgrowth potential in vitro, PBMC of healthy controls and transplant recipients with and without CTD were cultured under fibrotic culture conditions, and indeed a significant correlation (p=0.0002, r=0.62) was observed. Finally, double staining for α-SMA and CD16 revealed presence of α-SMA(+)CD16(+) cells in kidney explants from CTD patients, albeit at very low numbers. Our data represent evidence that, compared to CD14(++)CD16(-) monocytes, CD14(+)CD16(++) monocytes have an increased expression of smooth muscle cell-associated genes. This monocyte subpopulation is reduced in renal transplant patients with CTD, possibly due to selective migration into the allograft.