Controlled release of growth factors using synthetic glycosaminoglycans in a modular macroporous scaffold for tissue regeneration.

Healthy regeneration of tissue relies on a well-orchestrated release of growth factors. Herein, we show the use of synthetic glycosaminoglycans for controlled binding and release of growth factors to induce a desired cellular response. First, we screened glycosaminoglycans with growth factors of interest to determine kon (association rate constant), koff (dissociation rate constant), and Kd (equilibrium rate constant). As proof-of-concept, we functionalized an elastin-like recombinamer (ELR) hydrogel with a synthetic glycosaminoglycan and immobilized fibroblast growth factor 2 (FGF2), demonstrating that human umbilical vein endothelial cells cultured on top of ELR hydrogel differentiated into tube-like structures. Taking this concept further, we developed a tunable macroporous ELR cryogel material, containing a synthetic glycosaminoglycan and FGF2 that showed increased blood vessel formation and reduced immune response compared to control when implanted in a subcutaneous mouse model. These results demonstrated the possibility for specific release of desired growth factors in/from a modular 3D scaffold in vitro and in vivo.

Spatial interplay of lymphocytes and fibroblasts in estrogen receptor-positive HER2-negative breast cancer.

In estrogen-receptor-positive, HER2-negative (ER+HER2-) breast cancer, higher levels of tumor infiltrating lymphocytes (TILs) are often associated with a poor prognosis and this phenomenon is still poorly understood. Fibroblasts represent one of the most frequent cells in breast cancer and harbor immunomodulatory capabilities. Here, we evaluate the molecular and clinical impact of the spatial patterns of TILs and fibroblast in ER+HER2- breast cancer. We used a deep neural network to locate and identify tumor, TILs, and fibroblasts on hematoxylin and eosin-stained slides from 179 ER+HER2- breast tumors (ICGC cohort) together with a new density estimation analysis to measure the spatial patterns. We clustered tumors based on their spatial patterns and gene set enrichment analysis was performed to study their molecular characteristics. We independently assessed the spatial patterns in a second cohort of ER+HER2- breast cancer (N = 630, METABRIC) and studied their prognostic value. The spatial integration of fibroblasts, TILs, and tumor cells leads to a new reproducible spatial classification of ER+HER2- breast cancer and is linked to inflammation, fibroblast meddling, or immunosuppression. ER+HER2- patients with high TIL did not have a significant improved overall survival (HR = 0.76, P = 0.212), except when they had received chemotherapy (HR = 0.447). A poorer survival was observed for patients with high fibroblasts that did not show a high level of TILs (HR = 1.661, P = 0.0303). Especially spatial mixing of fibroblasts and TILs was associated with a good prognosis (HR = 0.464, P = 0.013). Our findings demonstrate a reproducible pipeline for the spatial profiling of TILs and fibroblasts in ER+HER2- breast cancer and suggest that this spatial interplay holds a decisive role in their cancer-immune interactions.

The effect of high-frequency electric pulses on tumor blood flow in vivo.

The aim of this study was to evaluate the effect of a 5-kHz repetition frequency of electroporating electric pulses in comparison to the standard 1-Hz frequency on blood flow of invasive ductal carcinoma tumors in Balb/C mice. Electroporation was performed by the delivery of eight electric pulses of 1,000 V cm(-1) and 100 mus duration at a repetition frequency of 1 Hz or 5 kHz. Blood flow changes in tumors were measured by laser Doppler flowmetry. Monitoring was performed continuously for 10 min before application of the electric pulses as well as immediately after application of the electric pulses for 40 min. The delivery of electric pulses to tumors induced changes in tumor blood flow. The reduction in blood flow started after the stimulation and continued for the 40-min period of observation. There was a significant difference in blood flow changes 3 min after application of the electric pulses at 1-Hz or 5-kHz repetition frequency. However, after 3 min the difference became nonsignificant. The findings showed that the high pulse frequency (5 kHz) had an effect comparable to the 1-Hz frequency on tumor blood flow except at very short times after pulse delivery, when pulses at 5 kHz produced a more intense reduction of blood flow.

Maturation of NMDA receptor-mediated spontaneous postsynaptic currents in the rat locus coeruleus neurons.

INTRODUCTION: During mammalian brain development, neural activity leads to maturation of glutamatergic innervations to locus coeruleus. In this study, fast excitatory postsynaptic currents mediated by N-methyl-d-aspartate (NMDA) receptors were evaluated to investigate the maturation of excitatory postsynaptic currents in locus coeruleus (LC) neurons. METHODS: NMDA receptor-mediated synaptic currents in LC neurons were evaluated using whole-cell voltage-clamp recording during the primary postnatal weeks. This technique was used to calculate the optimum holding potential for NMDA receptor-mediated currents and the best frequency for detecting spontaneous excitatory postsynaptic currents (sEPSC). RESULTS: The optimum holding potential for detecting NMDA receptor-mediated currents was + 40 to + 50 mV in LC neurons. The frequency, amplitude, rise time, and decay time constant of synaptic responses depended on the age of the animal and increased during postnatal maturation. CONCLUSION: These findings suggest that most nascent glutamatergic synapses express functional NMDA receptors in the postnatal coerulear neurons, and that the activities of the neurons in this region demonstrate an age-dependent variation.

Pyrroloquinoline quinone nutritional status alters lysine metabolism and modulates mitochondrial DNA content in the mouse and rat.

Pyrroloquinoline quinone (PQQ) added to purified diets devoid of PQQ improves indices of perinatal development in rats and mice. Herein, PQQ nutritional status and lysine metabolism are described, prompted by a report that PQQ functions as a vitamin-like enzymatic cofactor important in lysine metabolism (Nature 422 [2003] 832). Alternatively, we propose that PQQ influences lysine metabolism, but by mechanisms that more likely involve changes in mitochondrial content. PQQ deprivation in both rats and mice resulted in a decrease in mitochondrial content. In rats, alpha-aminoadipic acid (alphaAA), which is derived from alpha-aminoadipic semialdehyde (alphaAAS) and made from lysine in mitochondria, and the plasma levels of amino acids known to be oxidized in mitochondria (e.g., Thr, Ser, and Gly) were correlated with changes in the liver mitochondrial content of PQQ-deprived rats, but not PQQ-supplemented rats. In contrast, the levels of NAD dependent alpha-aminoadipate-delta-semialdehyde dehydrogenase (AASDH), a cytosolic enzyme important to alphaAA production from alphaAAS, was not influenced by PQQ dietary status. Moreover, the levels of U26 mRNA were not significantly changed even when diets differed markedly in PQQ and dietary lysine content. U26 mRNA levels were measured, because of U26's proposed, albeit questionable role as a PQQ-dependent enzyme involved in alphaAA formation.

Responsiveness of vascular alpha1-adrenoceptors of diabetic rat knee joint to phenylephrine in acute inflammation.

In diabetic angiopathy, responsiveness of alphal-adrenoceptors in blood vessels increases. The aim of this study was to investigate the vasoconstrictor response of knee joint blood vessels to phenylephrine (a 1-adrenoceptor agonist) in diabetes and acute inflammation. Acute knee joint inflammation was induced by the intraarticular injection of a 3% kaolin/3% carrageenan suspension. Diabetes was induced by the intravenous injection of alloxan (70 mg/kg). Male albino rats weighing 70 to 90 g each were divided into the following 4 groups: untreated controls, diabetic, inflammatory, and diabetic inflammatory. The blood flow of the knee joint was measured using the laser Doppler flowmetry (LDF) technique. Vasoconstriction of the articular microvascular was measured in response to the topical application of different concentrations (10(-7) to 10(-3) mol) phenylephrine. The results of this study show that (a) increased knee joint diameter and circumference due to inflammation and the knee joint basal blood flow were significantly lower in diabetic than in control rats; (b) the responsiveness of alphal-adrenoceptors decreased in kaolin/carrageenan-induced acute inflammation; (c) carrageenan-induced acute inflammation did not decrease the responsiveness of alphal-adrenoceptors in diabetic rats. We conclude that diabetes inhibits the reductive effect of acute inflammation on the responsiveness of alpha1-adrenoceptors in rats.

The ability of hippocampal CA1 area for induction of long-term potentiation is persistently reduced by prior treatment with cysteamine: an in vitro study.

Using field potential recording in the CA1 region of hippocampal slices from rats injected with cysteamine (200 mg/kg, s.c.), changes in activity and plasticity of Schaffer collateral-CA1 pyramidal cell synapses were examined. Extracellular field potential recording prior to and following either theta-pattern primed bursts (PBs), perfusion with low Mg(2+) or with high Ca(2+), indicated long-term potentiation (LTP) of population spikes amplitude (PSA). The extent of LTP of PSA was significantly lower in cysteamine-treated rats. It is concluded that cysteamine can entail lasting modifications in susceptibility of hippocampal CA1 for synaptic plasticity induced by tetanus. Similarly, disability in function of CA1 synapses can be traced by other protocols of LTP induction. The relevancy of the results to the facilitatory role of endogenous somatostatin in the function of Schaffer collateral-CA1 pyramidal cell synapses is also discussed.

Systemic naloxone enhances cerebral blood flow in anesthetized morphine-dependent rats.

Laser-Doppler flowmetry was used to study cerebral cortical blood flow responses to morphine and naloxone in morphine-naive and -dependent rats. The experiments were performed in spontaneously breathing anesthetized rats. Morphine (10 mg/kg, i.p.) administration reduced regional cerebral blood flow in control, sham-operated and morphine-dependent rats, but the depressant effect of morphine in morphine-dependent animals was less than that in control and sham-operated groups. While naloxone (0.5 mg/kg, s.c.) had no considerable effect on regional cerebral blood flow in control and sham-operated groups, it increased regional blood flow in morphine dependent ones. The depressant effect of morphine in all groups and the enhancing effect of naloxone in morphine-dependent animals were not seen after local application of lidocaine at the recording site. This study may provide a framework to study the cellular and molecular mechanisms responsible for coupling neuronal electrical activity with regional alterations in blood flow during precipitation of morphine withdrawal.

Esophageal cancer in Iran; a population-based study regarding adequacy of cancer surgery and overall survival.

AIMS: Prognosis of esophageal cancer (EC) is poor. Population-based studies regarding EC survival and adequacy of cancer surgery (ACS) from developing countries are lacking. We aimed to evaluate EC patients' survival and ACS in a population-based setting for the first time from Iran. METHODS: We randomly selected 409 cases from all 3048 newly diagnosed EC patients who were registered in the nationwide cancer registry in calendar year 2005-2006 and followed them until 2009. An expert panel in Cancer Institute of Iran reviewed adequacy of esophageal cancer surgery on a national basis. RESULTS: Overall three-year survival rate was 17%. The median (95% CI) survival time of all patients and cases who underwent surgery alone were 8.5 (6.9-10.1) and 6.0 (3.1-8.9) months, respectively. The hospital mortality was 27.8% among the operated patients. Only 6.8% of cases had documented free circumferential resection margin and merely 4.0% of patients experienced resection of more than 15 lymph nodes during surgery. CONCLUSIONS: In Iran, the overall survival of EC patients with different treatment modalities are slightly less than those reported from high-income countries, but cases with surgery alone as the main treatment had worse outcome compared with their counterparts in developed countries. Surgery with curative intent has not been performed in most cases in Iran. It seems current clinical outcome of EC patients could be improved with surgical quality improvement and/or more use of chemoradiation in Iran.

Frequent MYC coamplification and DNA hypomethylation of multiple genes on 8q in 8p11-p12-amplified breast carcinomas.

Genetic and epigenetic (DNA methylation, histone modifications, microRNA expression) crosstalk promotes inactivation of tumor suppressor genes or activation of oncogenes by gene loss/hypermethylation or duplications/hypomethylation, respectively. The 8p11-p12 chromosomal region is a hotspot for genomic aberrations (chromosomal rearrangements, amplifications and deletions) in several cancer forms, including breast carcinoma where amplification has been associated with increased proliferation rates and reduced patient survival. Here, an integrative genomics screen (DNA copy number, transcriptional and DNA methylation profiling) performed in 229 primary invasive breast carcinomas identified substantial coamplification of the 8p11-p12 genomic region and the MYC oncogene (8q24.21), as well as aberrant methylation and transcriptional patterns for several genes spanning the 8q12.1-q24.22 genomic region (ENPP2, FABP5, IMPAD1, NDRG1, PLEKHF2, RRM2B, SQLE, TAF2, TATDN1, TRPS1, VPS13B). Taken together, our findings suggest that MYC activity and aberrant DNA methylation may also have a pivotal role in the aggressive tumor phenotype frequently observed in breast carcinomas harboring 8p11-p12 regional amplification.

Decreased response of rat knee joint blood vessels to phenylephrine in chronic inflammation: involvement of nitric oxide.

The effect of chronic inflammation induced by Freund's Complete Adjuvant (FCA) on rat articular blood vessels and knee joint diameter was investigated. Blood flow changes in response to phenylephrine (an 1-adrenoceptor agonist) in FCA-treated and contralateral knee joints were studied over a 40 day period, using the laser Doppler flowmetery (LDF) technique. Unilateral injection of FCA (0.2 ml) increased the injected knee diameter on all days examined post-injection (P < 0.001) and its maximum increase (53 +/- 2 %) was reached on day 3. After this, the diameter decreased gradually but did not return to its initial value. In control animals, topical application of 10-13-10- 7 mol phenylephrine onto the exposed joint capsule decreased blood flow dose dependently (11. 1 +/- 4.4 to 58.2 +/- 4.5 %, respectively, P < 0.001). Unilateral injection with FCA attenuated the phenylephrine response in both ipsilateral and contralateral knees compared with the response of control animals (5.2 +/- 1.6 to 48.3 +/- 6.1 % and 1.9 +/- 2.2 to 45. 3 +/- 5.6 %, respectively, P < 0.05). The reduction persisted for 3 weeks after FCA injection (ipsilateral for 21 days; contralateral for 30 days, P < 0.001). Subsequently the response returned towards normal. To avoid the influence of 2-adrenoceptors, yohimbine (an 2-adrenoceptor antagonist) was injected (0.5 mg kg-1, I.P.) 30 min before phenylephrine application. Yohimbine blocked the vasoconstrictor effect of 10-10-10-7 mol clonidine (an 2-adrenoceptor agonist, topical application) by 44-67.7 % inhibition, respectively (P < 0.001). Prazosin (an 1-adrenoceptor antagonist, 0.1 mg kg-1, I.P.) blocked the vasoconstrictor effect of phenylephrine (10-10-10-7 mol, topical application) effectively (42 to 69.8 % inhibition, respectively, P < 0.001). To assess the role of nitric oxide (NO) on the observed responses, N G-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) was applied topically (0.2 micromol) 5 min before phenylephrine application. L-NAME application at 7 and 14 days after FCA injection potentiated the vasoconstrictor response in the FCA-treated knee (P < 0.001) but had no significant effect on the contralateral knee. Blood pressure monitoring during phenylephrine, clonidine and L-NAME administration indicated that topical application of the drugs had no significant effect on the systemic blood pressure. These findings indicate that the vasoconstrictor response to phenylephrine was decreased in chronic inflammation and increased NO production could be involved.