KLK6 expression in skin induces PAR1-mediated psoriasiform dermatitis and inflammatory joint disease.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation via cleavage of protease-activated receptor 1 (PAR1) and PAR2. KLK6 levels are elevated in multiple inflammatory and autoimmune conditions, but no definitive role in pathogenesis has been established. Here, we show that skin-targeted overexpression of KLK6 causes generalized, severe psoriasiform dermatitis with spontaneous development of debilitating psoriatic arthritis-like joint disease. The psoriatic skin and joint phenotypes are reversed by normalization of skin KLK6 levels and attenuated following genetic elimination of PAR1 but not PAR2. Conservation of this regulatory pathway was confirmed in human psoriasis using vorapaxar, an FDA-approved PAR1 antagonist, on explanted lesional skin from patients with psoriasis. Beyond defining a critical role for KLK6/PAR1 signaling in promoting psoriasis, our results demonstrate that KLK6/PAR1-mediated inflammation in the skin alone is sufficient to drive inflammatory joint disease. Further, we identify PAR1 as a promising cytokine-independent target in therapy of psoriasis and psoriatic arthritis.

Synergistic combination treatment to break cross talk between cancer cells and bone cells to inhibit progression of bone metastasis.

Advanced-stage cancers often metastasize to bone, and is the major cause of cancer-related morbidity and mortality. Due to poor biodistribution of intravenously administered anticancer drugs within the bone, chemotherapy is not optimally effective in treating bone metastasis. Additionally, overexpression of receptor activator of nuclear factor κB ligand (RANKL) in the bone microenvironment drives the vicious, destructive cycle of progression of bone metastasis and bone resorption. We hypothesized that the combination treatment - with docetaxel (TXT), an anticancer drug encapsulated in sustained release biodegradable nanoparticles (TXT-NPs) that are designed to localize in bone marrow, and denosumab monoclonal antibody (DNmb), which binds to RANKL - could be more effective than either treatment alone. We tested our hypothesis in intraosseous prostate cancer (PC-3) cell-induced osteolytic mouse model of bone metastasis with treatments given intravenously. The results demonstrated better efficacy with TXT-NPs than with TXT-CrEL or saline control in inhibiting progression of metastasis and improving survival. TXT-NPs showed ~3-fold higher drug levels in metastasized bone tissue at 1 wk post-administration than TXT-CrEL, thus explaining their efficacy. However, the combination treatment (TXT-NPs + DNmb) given simultaneously was significantly more effective in inhibiting metastatic progression; it caused early tumor regression and improved survival, and caused no body weight loss or tumor relapse, even when the treatment was discontinued, whereas TXT-NPs or DNmb alone treatments showed tumor relapse after an initial regression. Micro-CT analysis of the bone from the combination treatment showed no bone loss and normal bone mineral content, bone density, and bone volume fraction, whereas TXT-NPs or DNmb alone treatments showed bone loss. Confirming the above results, histochemical analysis of the bone from the combination treatment demonstrated normal bone morphology, and osteoblast and osteoclast cell activities. In conclusion, TXT-NPs and DNmb in combination, because of their complementary roles in breaking the cross talk between cancer cells and bone cells, was significantly effective in treating bone metastasis.

Ciliary genes arl13b, ahi1 and cc2d2a differentially modify expression of visual acuity phenotypes but do not enhance retinal degeneration due to mutation of cep290 in zebrafish.

Mutations in the gene Centrosomal Protein 290 kDa (CEP290) result in multiple ciliopathies ranging from the neonatal lethal disorder Meckel-Gruber Syndrome to multi-systemic disorders such as Joubert Syndrome and Bardet-Biedl Syndrome to nonsyndromic diseases like Leber Congenital Amaurosis (LCA) and retinitis pigmentosa. Results from model organisms and human genetics studies, have suggest that mutations in genes encoding protein components of the transition zone (TZ) and other cilia-associated proteins can function as genetic modifiers and be a source for CEP290 pleiotropy. We investigated the zebrafish cep290fh297/fh297 mutant, which encodes a nonsense mutation (p.Q1217*). This mutant is viable as adults, exhibits scoliosis, and undergoes a slow, progressive cone degeneration. The cep290fh297/fh297 mutants showed partial mislocalization of the transmembrane protein rhodopsin but not of the prenylated proteins rhodopsin kinase (GRK1) or the rod transducin subunit GNB1. Surprisingly, photoreceptor degeneration did not trigger proliferation of Müller glia, but proliferation of rod progenitors in the outer nuclear layer was significantly increased. To determine if heterozygous mutations in other cilia genes could exacerbate retinal degeneration, we bred cep290fh297/fh297 mutants to arl13b, ahi1, and cc2d2a mutant zebrafish lines. While cep290fh297/fh297 mutants lacking a single allele of these genes did not exhibit accelerated photoreceptor degeneration, loss of one alleles of arl13b or ahi1 reduced visual performance in optokinetic response assays at 5 days post fertilization. Our results indicate that the cep290fh297/fh297 mutant is a useful model to study the role of genetic modifiers on photoreceptor degeneration in zebrafish and to explore how progressive photoreceptor degeneration influences regeneration in adult zebrafish.

Differential Adaptations of the Musculoskeletal System after Spinal Cord Contusion and Transection in Rats.

Spinal cord injury (SCI) causes impaired neuronal function with associated deficits in the musculoskeletal system, which can lead to permanent disability. Here, the impact of SCI on in vivo musculoskeletal adaptation was determined by studying deficits in locomotor function and analyzing changes that occur in the muscle and bone compartments within the rat hindlimb after contusion or transection SCI. Analyses of locomotor patterns, as assessed via the Basso, Beattie, and Bresnahan (BBB) rating scale, revealed that transection animals showed significant deficits, while the contusion group had moderate deficits, compared with naïve groups. Muscle myofiber cross-sectional areas (CSA) of both the soleus and tibialis anterior muscles were significantly decreased three months after contusion SCI. Such decreases in CSA were even more dramatic in the transection SCI group, suggesting a dependence on muscle activity, which is further validated by the correlation analyses between BBB score and myofiber CSA. Bone compartment analyses, however, revealed that transection animals showed the most significant deficits, while contusion animals showed no significant differences in the trabecular bone content within the proximal tibia compartment. In general, values of bone volume per total bone volume (BV/TV) were similar across the SCI groups. Significant decreases were observed, however, in the transection animals for bone mineral content, bone mineral density, and three-dimensional trabecular structure parameters (trabecular number, thickness, and spacing) compared with the naïve and contusion groups. Together, these findings suggest an altered musculoskeletal system can be correlated directly to motor dysfunctions seen after SCI.

The Efficiency of Bone Marrow Aspiration for the Harvest of Connective Tissue Progenitors from the Human Iliac Crest.

BACKGROUND: The rational design and optimization of tissue engineering strategies for cell-based therapy requires a baseline understanding of the concentration and prevalence of osteogenic progenitor cell populations in the source tissues. The aim of this study was to (1) define the efficiency of, and variation among individuals in, bone marrow aspiration as a means of osteogenic connective tissue progenitor (CTP-O) harvest compared with harvest from iliac cancellous bone, and (2) determine the location of CTP-Os within native cancellous bone and their distribution between the marrow-space and trabecular-surface tissue compartments. METHODS: Eight 2-mL bone marrow aspiration (BMA) samples and one 7-mm transcortical biopsy sample were obtained from the anterior iliac crest of 33 human subjects. Two cell populations were obtained from the iliac cancellous bone (ICB) sample. The ICB sample was placed into αMEM (alpha-minimal essential medium) with antibiotic-antimycotic and minced into small pieces (1 to 2 mm in diameter) with a sharp osteotome. Cells that could be mechanically disassociated from the ICB sample were defined as marrow-space (IC-MS) cells, and cells that were disassociated only after enzymatic digestion were defined as trabecular-surface (IC-TS) cells. The 3 sources of bone and marrow-derived cells were compared on the basis of cellularity and the concentration and prevalence of CTP-Os through colony-forming unit (CFU) analysis. RESULTS: Large variation was seen among patients with respect to cell and CTP-O yield from the IC-MS, IC-TS, and BMA samples and in the relative distribution of CTP-Os between the IC-MS and IC-TS fractions. The CTP-O prevalence was highest in the IC-TS fraction, which was 11.4-fold greater than in the IC-MS fraction (p < 0.0001) and 1.7-fold greater than in the BMA fraction. However, the median concentration of CTP-Os in the ICB (combining MS and TS fractions) was only 3.04 ± 1.1-fold greater than that in BMA (4,265 compared with 1,402 CTP/mL; p = 0.00004). CONCLUSIONS: Bone marrow aspiration of a 2-mL volume at a given needle site is an effective means of harvesting CTP-Os, albeit diluted with peripheral blood. However, the median concentration of CTP-Os is 3-fold less than from native iliac cancellous bone. The distribution of CTP-Os between the IC-MS and IC-TS fractions varies widely among patients. CLINICAL RELEVANCE: Bone marrow aspiration is an effective means of harvesting CTP-Os but is associated with dilution with peripheral blood. Overall, we found that 63.5% of all CTP-Os within iliac cancellous bone resided on the trabecular surface; however, 48% of the patients had more CTP-Os contributed by the IC-MS than the IC-TS fraction.

The influence of tethered epidermal growth factor on connective tissue progenitor colony formation.

Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation.