Background Lymphatic vessels interconnect with blood vessels to form an elaborate system that aids in the control of tissue pressure and edema formation. Although the lymphatic system has been known to exist in a heart, little is known about the role the cardiac lymphatic system plays in the development of heart failure. Methods and Results Mice (C57 BL /6J, male, 8 to 12 weeks of age) were subjected to either myocardial ischemia or myocardial ischemia and reperfusion for up to 28 days. Analysis revealed that both models increased the protein expression of vascular endothelial growth factor C and VEGF receptor 3 starting at 1 day after the onset of injury, whereas a significant increase in lymphatic vessel density was observed starting at 3 days. Further studies aimed to determine the consequences of inhibiting the endogenous lymphangiogenesis response on the development of heart failure. Using 2 different pharmacological approaches, we found that inhibiting VEGF receptor 3 with MAZ ‐51 and blocking endogenous vascular endothelial growth factor C with a neutralizing antibody blunted the increase in lymphatic vessel density, blunted lymphatic transport, increased inflammation, increased edema, and increased cardiac dysfunction. Subsequent studies revealed that augmentation of the endogenous lymphangiogenesis response with vascular endothelial growth factor C treatment reduced inflammation, reduced edema, and improved cardiac dysfunction. Conclusions These results suggest that the endogenous lymphangiogenesis response plays an adaptive role in the development of ischemic‐induced heart failure and supports the emerging concept that therapeutic lymphangiogenesis is a promising new approach for the treatment of cardiovascular disease.

Together, these results suggest that hydrogen sulfide is an important regulator of cardiac mitochondrial content and establishes that exogenous hydrogen sulfide can induce mitochondrial biogenesis via an AMPK-PGC1α signaling cascade.

Neural adhesion proteins are crucial in the development and maintenance of functional neural connectivity. Growing evidence suggests that the IgLON family of neural adhesion molecules LSAMP, NTM, NEGR1, and OPCML are important candidates in forming the susceptibility to schizophrenia (SCZ). IgLON proteins have been shown to be involved in neurite outgrowth, synaptic plasticity and neuronal connectivity, all of which have been shown to be altered in the brains of patients with the diagnosis of schizophrenia. Here we optimized custom 5′-isoform-specific TaqMan gene-expression analysis for the transcripts of human IgLON genes to study the expression of IgLONs in the dorsolateral prefrontal cortex (DLPFC) of schizophrenic patients (n = 36) and control subjects (n = 36). Uniform 5′-region and a single promoter was confirmed for the human NEGR1 gene by in silico analysis. IgLON5, a recently described family member, was also included in the study. We detected significantly elevated levels of the NEGR1 transcript (1.33-fold increase) and the NTM 1b isoform transcript (1.47-fold increase) in the DLPFC of schizophrenia patients compared to healthy controls. Consequent protein analysis performed in male subjects confirmed the increase in NEGR1 protein content both in patients with the paranoid subtype and in patients with other subtypes. In-group analysis of patients revealed that lower expression of certain IgLON transcripts, mostly LSAMP 1a and 1b, could be related with concurrent depressive endophenotype in schizophrenic patients. Additionally, our study cohort provides further evidence that cannabis use may be a relevant risk factor associated with suicidal behaviors in psychotic patients. In conclusion, we provide clinical evidence of increased expression levels of particular IgLON family members in the DLPFC of schizophrenic patients. We propose that alterations in the expression profile of IgLON neural adhesion molecules are associated with brain circuit disorganization in neuropsychiatric disorders, such as schizophrenia. In the light of previously published data, we suggest that increased level of NEGR1 in the frontal cortex may serve as molecular marker for a wider spectrum of psychiatric conditions.

Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.

Highlights d Proline synthesis through PYCR1 is increased in low-oxygen conditions d PYCR1 activity in hypoxia supports TCA cycle function through NADH oxidation d PYCR1 is required for maintenance of hypoxic tumor regions

IgLON family is composed of five genes: Lsamp, Ntm, Opcml, Negr1, and Iglon5; encoding for five highly homologous neural adhesion proteins that regulate neurite outgrowth and synapse formation. In the current study we performed in silico analysis revealing that Ntm and Opcml display similar genomic structure as previously reported for Lsamp, characterized by two alternative promotors 1a and 1b. Negr1 and Iglon5 transcripts have uniform 5′ region, suggesting single promoter. Iglon5, the recently characterized family member, shares high level of conservation and structural qualities characteristic to IgLON family such as N-terminal signal peptide, three Ig domains, and GPI anchor binding site. By using custom 5′-isoform-specific TaqMan gene-expression assay, we demonstrated heterogeneous expression of IgLON transcripts in different areas of mouse brain and several-fold lower expression in selected tissues outside central nervous system. As an example, the expression of IgLON transcripts in urogenital and reproductive system is in line with repeated reports of urogenital tumors accompanied by mutations in IgLON genes. Considering the high levels of intra-family homology shared by IgLONs, we investigated potential compensatory effects at the level of IgLON isoforms in the brains of mice deficient of one or two family members. We found that the lack of IgLONs is not compensated by a systematic quantitative increase of the other family members. On the contrary, the expression of Ntm 1a transcript and NEGR1 protein was significantly reduced in the frontal cortex of Lsamp-deficient mice suggesting that the expression patterns within IgLON family are balanced coherently. The actions of individual IgLONs, however, can be antagonistic as demonstrated by differential expression of Syp in deletion mutants of IgLONs. In conclusion, we show that the genomic twin-promoter structure has impact on both anatomical distribution and intra-family interactions of IgLON family members. Remarkable variety in the activity levels of 1a and 1b promoters both in the brain and in other tissues, suggests complex functional regulation of IgLONs by alternative signal peptides driven by 1a and 1b promoters.

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Mild hypothermia (32 °C) is routinely used in medical practice to alleviate hypoxic ischemic damage, however, the mechanisms that underlie its protective effects remain uncertain. Using a systems approach based on genome-wide expression screens, reporter assays and biochemical studies, we find that cellular hypothermia response is associated with the augmentation of major stress-inducible transcription factors Nrf2 and HIF1Α affecting the antioxidant system and hypoxia response pathways, respectively. At the same time, NF-κB, a transcription factor involved in the control of immune and inflammatory responses, was not induced by hypothermia. Furthermore, mild hypothermia did not trigger unfolded protein response. Lower temperatures (27 °C and 22 °C) did not activate Nrf2 and HIF1A pathways as efficiently as mild hypothermia. Current findings are discussed in the context of the thermodynamic hypothesis of therapeutic hypothermia. We argue that the therapeutic effects are likely to stem both from metabolic suppression (inhibitory component) and augmentation of stress tolerance (activating component). We argue that systems coping with cellular stressors are plausible targets of therapeutic hypothermia and deserve more attention in clinical hypothermia research.