Further evidence of the involvement of the Wnt signaling pathway in Dupuytren's disease

Genetic background plays an important role in the development of Dupuytren's disease. A genome-wide association study (GWAS) showed that nine loci are associated with the disease, six of which contain genes that are involved in Wnt signaling (WNT2, WNT4, WNT7B, RSPO2, SFRP4, SULF1). To obtain insight in the role of these genes, we performed expression studies on affected and unaffected patient's tissues. Surgically obtained nodules and cords from eight Dupuytren's patients were compared to patient-matched control tissue (unaffected transverse palmar fascia). The Wnt-related genes found in the GWAS, the classical Wnt-downstream protein beta-catenin, as well as (myo) fibroblast markers were analyzed using real-time qPCR and immunohistochemical stainings for mRNA levels and protein levels, respectively. The collagen-coding genes COL1A1 and COL3A1 were highly upregulated on mRNA level, both in cords and nodules. Three Wnt-related genes were found to be differently regulated compared to control tissue: WNT2 was downregulated in nodules, WNT7B was upregulated in nodules, and SFRP4 was upregulated in nodules and cords. Immunohistochemistry revealed significantly less staining of Wnt2 in cords, but significantly more staining for Wnt7b in nodules. There was significantly more staining of alpha-SMA in nodules and cord and beta-catenin in nodules than in control tissue. We found differences in expression, both at mRNA and protein level, in several Wnt-related genes found earlier to be associated with Dupuytren's disease. Of these, Wnt7b was upregulated and found in close association with both alpha-SMA and beta-catenin expressing cells, making it a candidate pro-fibrotic mediator in Dupuytren's disease

Matrix and cell phenotype differences in Dupuytren's disease

BACKGROUND: Dupuytren's disease is a fibroproliferative disease of the hand and fingers, which usually manifests as two different phenotypes within the same patient. The disease first causes a nodule in the palm of the hand, while later, a cord develops, causing contracture of the fingers. RESULTS: We set out to characterize the two phenotypes by comparing matched cord and nodule tissue from ten Dupuytren's patients. We found that nodule tissue contained more proliferating cells, CD68-positive macrophages and α-smooth muscle actin (α-SMA)-positive myofibroblastic cells. qPCR analysis showed an increased expression of COL1A1, COL1A2, COL5A1, and COL6A1 in nodule tissue compared to cord tissue. Immunohistochemistry showed less deposition of collagen type I in nodules, although they contained more fibronectin, collagen type V, and procollagen 1. Lower collagen levels in nodule were confirmed by HPLC measurements of the Hyp/Pro ratio. PCOLCE2, an activator of BMP1, the main enzyme cleaving the C-terminal pro-peptide from procollagen, was also reduced in nodule. Cord tissue not only contained more collagen I, but also higher levels of hydroxylysylpyridinoline and lysylpyridinoline residues per triple helix, indicating more crosslinks. CONCLUSIONS: Our results clearly show that in Dupuytren's disease, the nodule is the active disease unit, although it does not have the highest collagen protein levels. The difference in collagen type I deposition compared to mRNA levels and procollagen 1 levels may be connected to a decrease in procollagen processing

Microsphere-Based Rapamycin Delivery, Systemic Versus Local Administration in a Rat Model of Renal Ischemia/Reperfusion Injury

The increasing prevalence and treatment costs of kidney diseases call for innovative therapeutic strategies that prevent disease progression at an early stage. We studied a novel method of subcapsular injection of monodisperse microspheres, to use as a local delivery system of drugs to the kidney. We generated placebo- and rapamycin monodisperse microspheres to investigate subcapsular delivery of drugs. Using a rat model of acute kidney injury, subcapsular injection of placebo and rapamycin monodisperse microspheres (monospheres) was compared to subcutaneous injection, mimicking systemic administration. We did not find any adverse effects related to the delivery method. Irrespective of the injection site, a similar low dose of rapamycin was present in the circulation. However, only local intrarenal delivery of rapamycin from monospheres led to decreased macrophage infiltration and a significantly lower amount of myofibroblasts in the kidney, where systemic administration did not. Local delivery of rapamycin did cause a transient increase in the deposition of collagen I, but not of collagen III. We conclude that therapeutic effects can be increased when rapamycin is delivered subcapsularly by monospheres, which, combined with low systemic concentrations, may lead to an effective intrarenal delivery method

Reduction of Fibrogenesis by Selective Delivery of a Rho Kinase Inhibitor to Hepatic Stellate Cells in Mice

One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] has been shown to reduce fibrosis in animal models. However, kinase expression is ubiquitous, so any inhibitor may affect many cell types. We hypothesize that cell-specific delivery of a kinase inhibitor will be beneficial. Therefore, we conjugated Y27632 to the carrier mannose-6-phosphate (M6P) human serum albumin (HSA), which is taken up specifically in activated HSC through the M6P/insulin-like growth factor II receptor. This conjugate decreased protein expression of phosphorylated myosin light chain 2 (pMLC2) and vinculin, downstream of Rho kinase, in activated primary HSC and decreased the migration and contraction of HSC. In an ex vivo model, free Y27632 decreased contractility of rat aortas, whereas the Y27-conjugate did not, showing that the Y27-conjugate does not affect nontarget tissue. In chronic CCl(4)-induced liver fibrosis, both free drug and conjugate reduced HSC activation; however, only the Y27-conjugate significantly reduced collagen deposition. Treatment with the Y27-conjugate, but not with free drug, reduced pMLC2 expression in livers 24 h after injection, demonstrating prolonged inhibition of the Rho kinase pathway. The Rho kinase inhibitor Y27632 can be specifically targeted to HSC using M6PHSA, decreasing its effects in nontarget tissues. The targeted drug effectively reduced fibrotic parameters in vivo via the inhibition of the Rho kinase pathway

Increased Liver Uptake and Reduced Hepatic Stellate Cell Activation with a Cell-Specific Conjugate of the Rho-kinase Inhibitor Y27632

Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, but the ubiquitous presence of this kinase may hinder examination of its exact role and the therapeutic use of inhibitors. We therefore coupled the Rho-kinase inhibitor Y27632 to a drug carrier that binds the mannose-6-phosphate insulin-like growth factor II (M6P/IGFII)-receptor which is upregulated on activated HSC. Y27632 was coupled to mannose-6-phosphate human serum albumin (M6PHSA), and in vitro experiments were performed on primary rat HSC. Biodistribution and effect studies were performed in an acute CCl(4) model in mice. Y27-conjugate remained stable in serum, while drug was efficiently released in liver homogenates. Receptor-blocking studies revealed that it was specifically taken up through the M6P/IGFII-receptor on fibroblasts, and it inhibited expression of fibrotic markers in activated HSC. In vivo, liver drug levels were significantly higher after injection of Y27-conjugate as compared to Y27632, and the conjugate accumulated specifically in HSC. After acute CCl(4)-induced liver injury, Y27-conjugate reduced the local activation of HSC, whereas an equimolar dose of free drug did not. We conclude that specific targeting of a Rho-kinase inhibitor to HSC leads to enhanced accumulation of the drug in HSC, reducing early fibrogenesis in the liver

Further evidence of the involvement of the Wnt signaling pathway in Dupuytren’s disease

Genetic background plays an important role in the development of Dupuytren's disease. A genome-wide association study (GWAS) showed that nine loci are associated with the disease, six of which contain genes that are involved in Wnt signaling (WNT2, WNT4, WNT7B, RSPO2, SFRP4, SULF1). To obtain insight in the role of these genes, we performed expression studies on affected and unaffected patient's tissues. Surgically obtained nodules and cords from eight Dupuytren's patients were compared to patient-matched control tissue (unaffected transverse palmar fascia). The Wnt-related genes found in the GWAS, the classical Wnt-downstream protein beta-catenin, as well as (myo) fibroblast markers were analyzed using real-time qPCR and immunohistochemical stainings for mRNA levels and protein levels, respectively. The collagen-coding genes COL1A1 and COL3A1 were highly upregulated on mRNA level, both in cords and nodules. Three Wnt-related genes were found to be differently regulated compared to control tissue: WNT2 was downregulated in nodules, WNT7B was upregulated in nodules, and SFRP4 was upregulated in nodules and cords. Immunohistochemistry revealed significantly less staining of Wnt2 in cords, but significantly more staining for Wnt7b in nodules. There was significantly more staining of alpha-SMA in nodules and cord and beta-catenin in nodules than in control tissue. We found differences in expression, both at mRNA and protein level, in several Wnt-related genes found earlier to be associated with Dupuytren's disease. Of these, Wnt7b was upregulated and found in close association with both alpha-SMA and beta-catenin expressing cells, making it a candidate pro-fibrotic mediator in Dupuytren's disease

Glycosylation-mediated targeting of carriers

For safe and effective therapy, drugs should be delivered selectively to their target tissues or cells at an optimal rate. Drug delivery system technology maximizes the therapeutic efficacy and minimizes unfavorable drug actions by controlling their distribution profiles. Ligand-receptor binding is a typical example of specific recognition mechanisms in the body; therefore, ligand-modified drug carriers have been developed for active targeting based on receptor-mediated endocytosis. Among the various ligands reported thus far, sugar recognition is a promising approach for active targeting because of their high affinity and expression. Glycosylation has been applied for both macromolecular and liposomal carriers for cell-selective drug targeting. Recently, the combination of ultrasound exposure and glycosylated bubble liposomes has been developed. In this review, recent advances of glycosylation-mediated targeted drug delivery systems are discussed