Beyond PDE4 inhibition: A comprehensive review on downstream cAMP signaling in the central nervous system.

Cyclic adenosine monophosphate (cAMP) is a key second messenger that regulates signal transduction pathways pivotal for numerous biological functions. Intracellular cAMP levels are spatiotemporally regulated by their hydrolyzing enzymes called phosphodiesterases (PDEs). It has been shown that increased cAMP levels in the central nervous system (CNS) promote neuroplasticity, neurotransmission, neuronal survival, and myelination while suppressing neuroinflammation. Thus, elevating cAMP levels through PDE inhibition provides a therapeutic approach for multiple CNS disorders, including multiple sclerosis, stroke, spinal cord injury, amyotrophic lateral sclerosis, traumatic brain injury, and Alzheimer's disease. In particular, inhibition of the cAMP-specific PDE4 subfamily is widely studied because of its high expression in the CNS. So far, the clinical translation of full PDE4 inhibitors has been hampered because of dose-limiting side effects. Hence, focusing on signaling cascades downstream activated upon PDE4 inhibition presents a promising strategy, offering novel and pharmacologically safe targets for treating CNS disorders. Yet, the underlying downstream signaling pathways activated upon PDE(4) inhibition remain partially elusive. This review provides a comprehensive overview of the existing knowledge regarding downstream mediators of cAMP signaling induced by PDE4 inhibition or cAMP stimulators. Furthermore, we highlight existing gaps and future perspectives that may incentivize additional downstream research concerning PDE(4) inhibition, thereby providing novel therapeutic approaches for CNS disorders.

Development of Mesoporous Silica Nanoparticle-Based Films with Tunable Arginine-Glycine-Aspartate Peptide Global Density and Clustering Levels to Study Stem Cell Adhesion and Differentiation.

Stem cell adhesion is mediated via the binding of integrin receptors to adhesion motifs present in the extracellular matrix (ECM). The spatial organization of adhesion ligands plays an important role in stem cell integrin-mediated adhesion. In this study, we developed a series of biointerfaces using arginine-glycine-aspartate (RGD)-functionalized mesoporous silica nanoparticles (MSN-RGD) to study the effect of RGD adhesion ligand global density (ligand coverage over the surface), spacing, and RGD clustering levels on stem cell adhesion and differentiation. To prepare the biointerface, MSNs were chemically functionalized with RGD peptides via an antifouling poly(ethylene glycol) (PEG) linker. The RGD surface functionalization ratio could be controlled to create MSNs with high and low RGD ligand clustering levels. MSN films with varying RGD global densities could be created by blending different ratios of MSN-RGD and non-RGD-functionalized MSNs together. A computational simulation study was performed to analyze nanoparticle distribution and RGD spacing on the resulting surfaces to determine experimental conditions. Enhanced cell adhesion and spreading were observed when RGD global density increased from 1.06 to 5.32 nmol cm-2 using highly clustered RGD-MSN-based films. Higher RGD ligand clustering levels led to larger cell spreading and increased formation of focal adhesions. Moreover, a higher RGD ligand clustering level promoted the expression of alkaline phosphatase in hMSCs. Overall, these findings indicate that both RGD global density and clustering levels are crucial variables in regulating stem cell behaviors. This study provides important information about ligand-integrin interactions, which could be implemented into biomaterial design to achieve optimal performance of adhesive functional peptides.

Computational investigation of the dynamic control of cAMP signaling by PDE4 isoform types.

Cyclic adenosine monophosphate (cAMP) is a generic signaling molecule that, through precise control of its signaling dynamics, exerts distinct cellular effects. Consequently, aberrant cAMP signaling can have detrimental effects. Phosphodiesterase 4 (PDE4) enzymes profoundly control cAMP signaling and comprise different isoform types wherein enzymatic activity is modulated by differential feedback mechanisms. Because these feedback dynamics are non-linear and occur coincidentally, their effects are difficult to examine experimentally but can be well simulated computationally. Through understanding the role of PDE4 isoform types in regulating cAMP signaling, PDE4-targeted therapeutic strategies can be better specified. Here, we established a computational model to study how feedback mechanisms on different PDE4 isoform types lead to dynamic, isoform-specific control of cAMP signaling. Ordinary differential equations describing cAMP dynamics were implemented in the VirtualCell environment. Simulations indicated that long PDE4 isoforms exert the most profound control on oscillatory cAMP signaling, as opposed to the PDE4-mediated control of single cAMP input pulses. Moreover, elevating cAMP levels or decreasing PDE4 levels revealed different effects on downstream signaling. Together these results underline that cAMP signaling is distinctly regulated by different PDE4 isoform types and that this isoform specificity should be considered in both computational and experimental follow-up studies to better define PDE4 enzymes as therapeutic targets in diseases in which cAMP signaling is aberrant.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Reliability and Safety of Bedside Blind Bone Biopsy Performed by a Diabetologist for the Diagnosis and Treatment of Diabetic Foot Osteomyelitis.

OBJECTIVE: Bone biopsy (BB) performed by a surgeon or an interventional radiologist is recommended for suspicion of osteomyelitis underlying diabetic foot ulcer (DFU). To facilitate its practice, we developed a procedure allowing bedside blind bone biopsy (B4) by a diabetologist. RESEARCH DESIGN AND METHODS: We conducted a three-step observational study consisting of a feasibility and safety phase (phase 1) to assess the success and side effects of B4, a validity phase (phase 2) to compare DFU outcomes between positive (B4+) and negative (B4-) bone cultures, and a performance phase (phase 3) to compare B4 with the conventional surgical or radiological procedure basic bone biopsy (B3). Primary end points were the presence of bone tissue (phase 1) and complete DFU healing with exclusive medical treatment at 12 months (phases 2 and 3). RESULTS: In phase 1, 37 consecutive patients with clinical and/or radiological suspicion of DFU osteomyelitis underwent B4. Bone tissue was collected in all patients with few side effects. In phase 2, a B4+ bone culture was found in 40 of 79 (50.6%) participants. Among B4+ patients, complete wound healing after treatment was 57.5%. No statistical difference was observed with patients with B4- bone culture not treated with antibiotics (71.8%, P = 0.18). In phase 3, the proportion of patients with positive BB was lower in B4 (40 of 79, 50.6%) than in B3 (34 of 44, 77.3%, P < 0.01). However, complete healing was similar (64.6% vs. 54.6%, P = 0.28). No difference in rate of culture contamination was observed. CONCLUSIONS: B4 is a simple, safe, and efficient procedure for the diagnosis of DFU osteomyelitis with a similar proportion of healing to conventional BB.

Expanding Biomaterial Surface Topographical Design Space through Natural Surface Reproduction.

Surface topography is a tool to endow biomaterials with bioactive properties. However, the large number of possible designs makes it challenging to find the optimal surface structure to induce a specific cell response. The TopoChip platform is currently the largest collection of topographies with 2176 in silico designed microtopographies. Still, it is exploring only a small part of the design space due to design algorithm limitations and the surface engineering strategy. Inspired by the diversity of natural surfaces, it is assessed as to what extent the topographical design space and consequently the resulting cellular responses can be expanded using natural surfaces. To this end, 26 plant and insect surfaces are replicated in polystyrene and their surface properties are quantified using white light interferometry. Through machine-learning algorithms, it is demonstrated that natural surfaces extend the design space of the TopoChip, which coincides with distinct morphological and focal adhesion profiles in mesenchymal stem cells (MSCs) and Pseudomonas aeruginosa colonization. Furthermore, differentiation experiments reveal the strong potential of the holy lotus to improve osteogenesis in MSCs. In the future, the design algorithms will be trained with the results obtained by natural surface imprint experiments to explore the bioactive properties of novel surface topographies.

Diabetic foot ulcer management in a multidisciplinary foot centre: one-year healing, amputation and mortality rate.

OBJECTIVE: To describe the rates of healing, major amputation and mortality after 12 months in patients with a new diabetic foot ulcer (DFU) and their care in a French diabetic foot service (DFS). METHOD: A prospective single-centre study including patients from March 2009 to December 2010. The length of time to healing, minor amputation, major amputation and mortality rate after inclusion were analysed using the Kaplan-Meier method. RESULTS: Some 347 patients were included (3% lost to follow-up), with a median follow-up (IQR) of 19 (12-24) months. The mean (SD) age was 65±12 years, 68% were male, and the median duration of the ulcer was 49 (19-120) days. Complications of the DFU were ischaemia (70%), infection (55%) and osteomyelitis (47%). Of the patients, 50% were inpatients in the DFS at inclusion (median duration of hospitalisation 26 (15-41) days). The rate of healing at one year was 67% (95% confidence interval (CI): 61-72); of major amputation 10% (95% CI: 7-17); of minor amputation 19% (95% CI: 14-25), and the death rate was 9% (95% CI: 7-13). Using an adjusted hazard ratio, the predictive factors of healing were perfusion and the area of the wound. The risk factors for a major amputation were active smoking and osteomyelitis. The risk factors for mortality were perfusion and age. CONCLUSION: This study confirms the need to treat DFUs rapidly, in a multidisciplinary DFS.

On the correlation between material-induced cell shape and phenotypical response of human mesenchymal stem cells.

Learning rules by which cell shape impacts cell function would enable control of cell physiology and fate in medical applications, particularly, on the interface of cells and material of the implants. We defined the phenotypic response of human bone marrow-derived mesenchymal stem cells (hMSCs) to 2176 randomly generated surface topographies by probing basic functions such as migration, proliferation, protein synthesis, apoptosis, and differentiation using quantitative image analysis. Clustering the surfaces into 28 archetypical cell shapes, we found a very strict correlation between cell shape and physiological response and selected seven cell shapes to describe the molecular mechanism leading to phenotypic diversity. Transcriptomics analysis revealed a tight link between cell shape, molecular signatures, and phenotype. For instance, proliferation is strongly reduced in cells with limited spreading, resulting in down-regulation of genes involved in the G2/M cycle and subsequent quiescence, whereas cells with large filopodia are related to activation of early response genes and inhibition of the osteogenic process. In this paper we were aiming to identify a universal set of genes that regulate the material-induced phenotypical response of human mesenchymal stem cells. This will allow designing implants that can actively regulate cellular, molecular signalling through cell shape. Here we are proposing an approach to tackle this question.

Circulating Receptor Activator of Nuclear Factor kB Ligand and triglycerides are associated with progression of lower limb arterial calcification in type 2 diabetes: a prospective, observational cohort study.

BACKGROUND: Lower limb arterial calcification is a frequent, underestimated but serious complication of diabetes. The DIACART study is a prospective cohort study designed to evaluate the determinants of the progression of lower limb arterial calcification in 198 patients with type 2 diabetes. METHODS: Lower limb arterial calcification scores were determined by computed tomography at baseline and after a mean follow up of 31.20 ± 3.86 months. Serum RANKL (Receptor Activator of Nuclear factor kB Ligand) and bone remodeling, inflammatory and metabolic parameters were measured at baseline. The predictive effect of these markers on calcification progression was analyzed by a multivariate linear regression model. RESULTS: At baseline, mean ± SD and median lower limb arterial calcification scores were, 2364 ± 5613 and 527 respectively and at the end of the study, 3739 ± 6886 and 1355 respectively. Using multivariate analysis, the progression of lower limb arterial log calcification score was found to be associated with (ß coefficient [slope], 95% CI, p-value) baseline log(calcification score) (1.02, 1.00-1.04, p < 0.001), triglycerides (0.11, 0.03-0.20, p = 0.007), log(RANKL) (0.07, 0.02-0.11, p = 0.016), previous ischemic cardiomyopathy (0.36, 0.15-0.57, p = 0.001), statin use (0.39, 0.06-0.72, p = 0.023) and duration of follow up (0.04, 0.01-0.06, p = 0.004). CONCLUSION: In patients with type 2 diabetes, lower limb arterial calcification is frequent and can progress rapidly. Circulating RANKL and triglycerides are independently associated with this progression. These results open new therapeutic perspectives in peripheral diabetic calcifying arteriopathy. Trial registration NCT02431234.

Mechanotransduction is a context-dependent activator of TGF-ß signaling in mesenchymal stem cells.

We previously found that surface topographies induce the expression of the Scxa gene, encoding Scleraxis in tenocytes. Because Scxa is a TGF-ß responsive gene, we investigated the link between mechanotransduction and TGF-ß signaling. We discovered that mesenchymal stem cells exposed to both micro-topographies and TGF-ß2 display synergistic induction of SMAD phosphorylation and transcription of the TGF-ß target genes SCX, a-SMA, and SOX9. Pharmacological perturbations revealed that Rho/ROCK/SRF signaling is required for this synergistic response. We further found an activation of the early response genes SRF and EGR1 during the early adaptation phase on micro-topographies, which coincided with higher expression of the TGF-ß type-II receptor gene. Of interest, PKC activators Prostratin and Ingenol-3, known for inducing actin reorganization and activation of serum response elements, were able to mimic the topography-induced TGF-ß response. These findings provide novel insights into the convergence of mechanobiology and TGF-ß signaling, which can lead to improved culture protocols and therapeutic applications.

Diabetic foot ulcer management in a multidisciplinary foot centre: one-year healing, amputation and mortality rate.

OBJECTIVE: To describe the rates of healing, major amputation and mortality after 12 months in patients with a new diabetic foot ulcer (DFU) and their care in a French diabetic foot service (DFS). METHOD: A prospective single-centre study including patients from March 2009 to December 2010. The length of time to healing, minor amputation, major amputation and mortality rate after inclusion were analysed using the Kaplan-Meier method. RESULTS: Some 347 patients were included (3% lost to follow-up), with a median follow-up (IQR) of 19 (12-24) months. The mean (SD) age was 65±12 years, 68% were male, and the median duration of the ulcer was 49 (19-120) days. Complications of the DFU were ischaemia (70%), infection (55%) and osteomyelitis (47%). Of the patients, 50% were inpatients in the DFS at inclusion (median duration of hospitalisation 26 (15-41) days). The rate of healing at one year was 67% (95% confidence interval (CI): 61-72); of major amputation 10% (95% CI: 7-17); of minor amputation 19% (95% CI: 14-25), and the death rate was 9% (95% CI: 7-13). Using an adjusted hazard ratio, the predictive factors of healing were perfusion and the area of the wound. The risk factors for a major amputation were active smoking and osteomyelitis. The risk factors for mortality were perfusion and age. CONCLUSION: This study confirms the need to treat DFUs rapidly, in a multidisciplinary DFS.

Effect of ultrasound on bone fracture healing: A computational bioregulatory model.

Bone healing is a complex biological procedure in which several cellular actions, directed by biochemical and mechanical signals, take place. Experimental studies have shown that ultrasound accelerates bone ossification and has a multiple influence on angiogenesis. In this study a mathematical model predicting bone healing under the presence of ultrasound is demonstrated. The primary objective is to account for the ultrasound effect on angiogenesis and more specifically on the transport of the Vascular Endothelial Growth Factor (VEGF). Partial differential equations describing the spatiotemporal evolution of cells, growth factors, tissues and ultrasound acoustic pressure and velocity equations determining the development of the blood vessel network constitute the present model. The effect of the ultrasound characteristics on angiogenesis and bone healing is investigated by applying different boundary conditions of acoustic pressure at the periosteal region of the bone model, which correspond to different intensity values. The results made clear that ultrasound enhances angiogenesis mechanisms during bone healing. The proposed model could be regarded as a step towards the monitoring of the effect of ultrasound on bone regeneration.