Laminin-rich blood vessels display activated growth factor signaling and act as the proliferation centers in Dupuytren’s contracture

IntroductionDupuytren’s contracture (DC) is a chronic fibroproliferative disease of the hand, which is characterized by uncontrolled proliferation of atypical myofibroblasts at the cellular level. We hypothesized that specific areas of the DC tissue are sustaining the cell proliferation and studied the potential molecular determinants that might contribute to the formation of such niches.MethodsWe studied the expression pattern of cell proliferation marker Ki67, phosphorylated AKT (Ak mouse strain thymoma) kinase, DC-associated growth factors (connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 2 (IGF-2)) and extracellular matrix components (laminins, fibronectin, collagen IV) in DC tissue and normal palmar fascia using immunofluorescence microscopy and quantitative real-time polymerase chain reaction (qPCR).ResultsWe found that proliferative cells in the DC nodules were concentrated in the immediate vicinity of small blood vessels and localized predominantly in the myofibroblast layer. Correspondingly, the DC-associated blood vessels contained increased levels of phosphorylated AKT, a hallmark of activated growth factor signaling. When studying the expression of potential activators of AKT signaling we found that the expression of bFGF was confined to the endothelium of the small blood vessels, IGF-2 was present uniformly in the DC tissue and CTGF was expressed in the DC-associated sweat gland acini. In addition, the blood vessels in DC nodules contained increased amounts of laminins 511 and 521, which have been previously shown to promote the proliferation and stem cell properties of different cell types.ConclusionsBased on our findings, we propose that in the DC-associated small blood vessels the presence of growth factors in combination with favorable extracellular matrix composition provide a supportive environment for sustained proliferation of myofibroblasts and thus the blood vessels play an important role in DC pathogenesis.

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The online version of this article (doi:10.1186/s13075-015-0661-y) contains supplementary material, which is available to authorized users.     

Light- and CO2-saturated photosynthesis: enhancement by oxygen

Oxygen may enhance CO₂-saturated photosynthesis in intact leaves, which display the Warburg effect when illuminated at the current atmospheric level of CO₂ and O₂, of about 350 μl l⁻¹ and 21%, respectively. The magnitude of the stimulation depends on irradiance. The K _M(O₂) of the stimulation is 128 μM (10.6% O₂). Maximum enhancement in wheat leaves is 6.1 and 5.3 μmol m⁻² s⁻¹ under 27.9 and 18.7 mW cm⁻², respectively, corresponding to a 25–30% increase in the ribulose 1,5-bisphosphate (RuBP) turnover rate if compared with O₂-free ambient gas phase. The stimulation appears in 5–10 s after a sharp increase in O₂. In response to a decrease in O₂, the new stabilized rate is reached in 5–7 min. The stimulation does not involve any increase in the activity of Rubisco. The effect correlates with increased concentration of RuBP. Oxygen enhances CO₂-saturated photosynthesis by acting as a terminal electron acceptor in the photosynthetic electron transport. The magnitude of the effect may be adopted as an index of the pseudocyclic photophosphorylation in vivo.     

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo: Control by High CO2 Concentration

High CO₂ concentrations (HC) in air induce partial deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39). Under saturating irradiance, increase in [CO₂] to 1 200 cm³ m^–3 reduces the concentration of operating carboxylation centres by 20–30 %. At a further increase in [CO₂], the activity remained on the same level. Under limiting irradiance, the lowest activity was reached at 600 cm³(CO₂) m^–3. The presence of oxygen diminished deactivation, but O₂ failed to stimulate reactivation under high CO₂. Conditions that favour oxygenation of ribulose-1,5-bisphosphate (RuBP) facilitated reactivation. Even HC did not act as an inhibitor. HC induces deactivation of RuBPCO by increasing the concentration of free reaction centres devoid of the substrate, which are more vulnerable to inhibition than the centres filled with substrates or products.     

Estimation of rate constants of the partial reactions of carboxylation of ribulose-1,5-bisphosphate in vivo

An in vivo method for the estimation of kinetic parameters of partial reactions of carboxylation of ribulose 1,5-bisphosphate (RuBP) catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is described. Rubisco in barley, wheat and bean is different in the ability of its active centers to bind RuBP. The rate constant of the formation of the Rubisco-RuBP complex in these plants at 25 °C is 0.414, 0.245 and 0.660 mM^-1 s^-1, respectively. The rate constant of the reaction of the Rubisco-bound enediol with CO₂ does not differ significantly in barley and wheat, and averages 66 mM^-1 s^-1. Decreased irradiance inhibits Rubisco in two ways: by reducing the concentration of operating catalytic sites and by decreasing the rate constant of binding of RuBP to Rubisco. High concentrations of CO₂ inhibit Rubisco by decreasing the concentration of competent carboxylation centers, without any s ignificant influence upon the rate constants of partial reactions.     

Specificity factor of Rubisco: estimation in intact leaves by carboxylation at different CO2/O2 ratios

The specificity factor of Rubisco (S _f) was estimated in intact leaves from the carboxylation of ribulose-1,5-bisphosphate (RuBP) at various CO₂/O₂ ratios. As oxygenation is calculated by the difference of the ¹⁴CO₂ uptake by RuBP in the absence and presence of oxygen, it is important to choose the optimum CO₂/O₂ ratios. At high CO₂ concentration (1,000 cm³ m^?3 and higher) oxygenation consumes less than 50% RuBP but the difference of concentrations of CO₂ at cell walls (C _w) and at the carboxylation centers (C _c) is 2?C5% and the influence of mesophyll resistance (r _md) is of minor importance. To accumulate large endogenous pool of RuBP, the leaves were preilluminated in the CO₂- and O₂-free gas environments for 8 to 10 s. Thereafter the light was switched off and the leaves were flushed with the gas containing different concentrations of ¹⁴CO₂ and O₂. The specificity factor of Rubisco was calculated from the amount of the tracer taken up under different ¹⁴CO₂/O₂ ratios by the exhaustion of the RuBP pool. Application of ¹⁴CO₂ allowed us to discriminate between the CO₂ uptake and the concurrent respiratory CO₂ release which proceeded at the expense of unlabelled intermediates.     

Regulation of Rubisco: Influence of Parameters of Partial Reactions upon the Rate of Carboxylation/Oxygenation1

Activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) is regulated by environmental factors (irradiance, CO₂ concentration) by changing the concentration of competent reaction centers and the reactivity of the centers. These changes do not necessarily mean that the steady-state rate of carboxylation or oxygenation would change. A mathematical model of carboxylation/oxygenation has been developed to evaluate the significance of the regulation of particular paramaters for integrating the response.     

Predicting functional upstream open reading frames in Saccharomyces cerevisiae

Background  

Some upstream open reading frames (uORFs) regulate gene expression (i.e., they are functional) and can play key roles in keeping organisms healthy. However, how uORFs are involved in gene regulation is not yet fully understood. In order to get a complete view of how uORFs are involved in gene regulation, it is expected that a large number of experimentally verified functional uORFs are needed. Unfortunately, wet-experiments to verify that uORFs are functional are expensive.     

Measurement of the Concentration of the Active Centers of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo

Methods for in vivo measurement of the concentration of the reactive centers of ribulose-1,5,-bisphosphate carboxylase/oxygenase (Rubisco) are suggested that are based on saturation of the active centers with RuBP and determination of the concentration of the Rubisco–RuBP complex. The total concentration of potentially reactive centers is calculated from the dependence of the concentration of this complex on CO₂ concentration at a steady-state photosynthetic rate with further extrapolation of the carbon dioxide dependence curve to a zero CO₂ concentration. The concentration of centers that possessed a catalytic activity under given environmental conditions was measured after transferring leaves having a steady-state photosynthetic rate into a medium devoid of CO₂ and O₂. This procedure ensured the saturation of the carboxylation centers with RuBP. The carboxylation rates were measured during a short-term exposure to ¹⁴CO₂, and the concentration of the complex was calculated using the values of CO₂ concentration during the exposure time, as well as the carboxylation rate and constant. Rubisco activity was found to decrease at elevated CO₂ concentrations due to a lower concentration of catalytically active enzyme centers.