Mechanisms by which oxygen regulates gene expression and cell-cell interaction in the vasculature

Mechanisms by which oxygen regulates gene expression and cell-cell interaction in the vasculature. Hypoxia has profound effects on blood vessel tone. Acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia causes smooth muscle cell replication and extracellular matrix accumulation resulting in vessel wall remodeling. The cellular responses to hypoxia involve complex cell-cell interactions mediated by the release of growth factors, cytokines and biological messengers. We have reported that hypoxia increases the expression of a number of genes encoding vascular cell mitogens produced by endothelial cells: platelet-derived growth factor B(PDGF-B); endothelin-1 (ET-1); and vascular endothelial growth factor (VEGF). A 28-bp enhancer in the 5′ upstream region of the VEGF gene mediates the expression of VEGF by endothelial cells under conditions of hypoxia. Hypoxia, however, has opposite effects on the vasodilator nitric oxide (NO); hypoxia suppresses both the transcriptional rate of the endothelial nitric oxide synthase gene and the stability of its mRNA. These endothelial-dependent processes would lead to vessel wall remodeling characteristic of a number of diseases from atherosclerosis to pulmonary hypertension. The smooth muscle cell also responds to hypoxia. It increases the transcriptional rate of the heme oxygenase gene-1 responsible for the breakdown of heme to carbon monoxide (CO) and biliverdin. CO is a vasodilator with properties similar to the well-studied molecule NO. CO suppresses the production of ET-1 and PDGF-B by endothelial cells. The regulated production of NO and CO under hypoxia, therefore, results in complex feedback loop interactions leading to altered smooth muscle cell growth in an autocrine and paracrine manner

Perinatal Stress, Brain Inflammation and Risk of Autism-Review and Proposal

Background: Autism Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by varying deficits in social interactions, communication, and learning, as well as stereotypic behaviors. Despite the significant increase in ASD, there are few if any clues for its pathogenesis, hampering early detection or treatment. Premature babies are also more vulnerable to infections and inflammation leading to neurodevelopmental problems and higher risk of developing ASD. Many autism “susceptibility” genes have been identified, but “environmental” factors appear to play a significant role. Increasing evidence suggests that there are different ASD endophenotypes. Discussion: We review relevant literature suggesting in utero inflammation can lead to preterm labor, while insufficient development of the gut-blood–brain barriers could permit exposure to potential neurotoxins. This risk apparently may increase in parents with “allergic” or autoimmune problems during gestation, or if they had been exposed to stressors. The presence of circulating auto-antibodies against fetal brain proteins in mothers is associated with higher risk of autism and suggests disruption of the blood–brain-barrier (BBB). A number of papers have reported increased brain expression or cerebrospinal fluid (CSF) levels of pro-inflammatory cytokines, especially TNF, which is preformed in mast cells. Recent evidence also indicates increased serum levels of the pro-inflammatory mast cell trigger neurotensin (NT), and of extracellular mitochondrial DNA (mtDNA), which is immunogenic. Gene mutations of phosphatase and tensin homolog (PTEN), the negative regulator of the mammalian target of rapamycin (mTOR), have been linked to higher risk of autism, but also to increased proliferation and function of mast cells. Summary: Premature birth and susceptibility genes may make infants more vulnerable to allergic, environmental, infectious, or stress-related triggers that could stimulate mast cell release of pro-inflammatory and neurotoxic molecules, thus contributing to brain inflammation and ASD pathogenesis, at least in an endophenotype of ASD patients

Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency

Exosomes are defined as submicron (30–150 nm), lipid bilayer-enclosed extracellular vesicles (EVs), specifically generated by the late endosomal compartment through fusion of multivesicular bodies with the plasma membrane. Produced by almost all cells, exosomes were originally considered to represent just a mechanism for jettisoning unwanted cellular moieties. Although this may be a major function in most cells, evolution has recruited the endosomal membrane-sorting pathway to duties beyond mere garbage disposal, one of the most notable examples being its cooption by retroviruses for the generation of Trojan virions. It is, therefore, tempting to speculate that certain cell types have evolved an exosome subclass active in intracellular communication. We term this EV subclass “signalosomes” and define them as exosomes that are produced by the “signaling” cells upon specific physiological or environmental cues and harbor cargo capable of modulating the programming of recipient cells. Our recent studies have established that signalosomes released by mesenchymal stem/stromal cells (MSCs) represent the main vector of MSC immunomodulation and therapeutic action in animal models of lung disease. The efficacy of MSC-exosome treatments in a number of preclinical models of cardiovascular and pulmonary disease supports the promise of application of exosome-based therapeutics across a wide range of pathologies within the near future. However, the full realization of exosome therapeutic potential has been hampered by the absence of standardization in EV isolation, and procedures for purification of signalosomes from the main exosome population. This is mainly due to immature methodologies for exosome isolation and characterization and our incomplete understanding of the specific characteristics and molecular composition of signalosomes. In addition, difficulties in defining metrics for potency of exosome preparations and the challenges of industrial scale-up and good manufacturing practice compliance have complicated smooth and timely transition to clinical development. In this manuscript, we focus on cell culture conditions, exosome harvesting, dosage, and exosome potency, providing some empirical guidance and perspectives on the challenges in bringing exosome-based therapies to clinic

Molecular cloning and characterization of human endothelial nitric oxide synthase

AbstractThe constitutive calcium/calmodulin-dependent nitric oxide (NO) synthase expressed in vascular endothelium shares common biochemical and pharmacologic properties with neuronal NO synthase. However, recent cloning and molecular characterization of NO synthase from bovine endothelial cells indicated the existence of a family of constitutive NO synthases. Accordingly, we undertook molecular cloning and sequence analysis of human endothelial NO synthase. Complementary DNA clones predict a protein of 1,203 amino acids sharing 94% identity with the bovine endothelial protein, but only 60% identity with the rat brain NO synthase isoform. Northern blot analysis with an endothelial-derived cDNA identified a 4.6–4.8 kb mRNA transcript in HUVEC and in situ hybridization localized transcripts to vascular endothelium but not neuronal tissue

Divergent Cardiopulmonary Actions of Heme Oxygenase Enzymatic Products in Chronic Hypoxia

Hypoxia and pressure-overload induce heme oxygenase-1 (HO-1) in cardiomyocytes and vascular smooth muscle cells (VSMCs). HO-1(-/-) mice exposed to chronic hypoxia develop pulmonary arterial hypertension (PAH) with exaggerated right ventricular (RV) injury consisting of dilation, fibrosis, and mural thrombi. Our objective was to identify the HO-1 product(s) mediating RV protection from hypoxic injury in HO-1(-/-) mice.HO-1(-/-) mice were exposed to seven weeks of hypoxia and treated with inhaled CO or biliverdin injections. CO reduced right ventricular systolic pressure (RVSP) and prevented hypoxic pulmonary arteriolar remodeling in both HO-1(-/-) and control mice. Biliverdin had no significant effect on arteriolar remodeling or RVSP in either genotype. Despite this, biliverdin prevented RV failure in the hypoxic HO-1(-/-) mice (0/14 manifested RV wall fibrosis or thrombus), while CO-treated HO-1(-/-) mice developed RV insults similar to untreated controls. In vitro, CO inhibited hypoxic VSMC proliferation and migration but did not prevent cardiomyocyte death from anoxia-reoxygenation (A-R). In contrast, bilirubin limited A-R-induced cardiomyocyte death but did not inhibit VSMC proliferation and migration.CO and bilirubin have distinct protective actions in the heart and pulmonary vasculature during chronic hypoxia. Moreover, reducing pulmonary vascular resistance may not prevent RV injury in hypoxia-induced PAH; supporting RV adaptation to hypoxia and preventing RV failure must be a therapeutic goal

Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells

Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues

Current Status of Targeted Therapy for Advanced Renal Cell Carcinoma

The treatment of metastatic renal cell carcinoma (mRCC) has recently evolved from being predominantly cytokine-based treatment to the use of targeted agents, which include sorafenib, sunitinib, bevacizumab (plus interferon alpha [IFN-α]), temsirolimus, everolimus, pazopanib, and most recently, axitinib. Improved understanding of the molecular pathways implicated in the pathogenesis of RCC has led to the development of specific targeted therapies for treating the disease. In Korea, it has been 5 years since targeted therapy became available for mRCC. Thus, we now have broader and better therapeutic options at hand, leading to a significantly improved prognosis for patients with mRCC. However, the treatment of mRCC remains a challenge and a major health problem. Many questions remain on the efficacy of combination treatments and on the best methods for achieving complete remission. Additional studies are needed to optimize the use of these agents by identifying those patients who would most benefit and by elucidating the best means of delivering these agents, either in combination or as sequential single agents. Furthermore, numerous ongoing research activities aim at improving the benefits of the new compounds in the metastatic situation or their application in the early phase of the disease. This review introduces what is currently known regarding the fundamental biology that underlies clear cell RCC, summarizes the clinical evidence supporting the benefits of targeted agents in mRCC treatment, discusses survival endpoints used in pivotal clinical trials, and outlines future research directions

The effects of timing of fine needle aspiration biopsies on gene expression profiles in breast cancers

<p>Abstract</p> <p>Background</p> <p>DNA microarray analysis has great potential to become an important clinical tool to individualize prognostication and treatment for breast cancer patients. However, with any emerging technology, there are many variables one must consider before bringing the technology to the bedside. There are already concerted efforts to standardize protocols and to improve reproducibility of DNA microarray. Our study examines one variable that is often overlooked, the timing of tissue acquisition, which may have a significant impact on the outcomes of DNA microarray analyses especially in studies that compare microarray data based on biospecimens taken <it>in vivo </it>and <it>ex vivo</it>.</p> <p>Methods</p> <p>From 16 patients, we obtained paired fine needle aspiration biopsies (FNABs) of breast cancers taken before (PRE) and after (POST) their surgeries and compared the microarray data to determine the genes that were differentially expressed between the FNABs taken at the two time points. qRT-PCR was used to validate our findings. To examine effects of longer exposure to hypoxia on gene expression, we also compared the gene expression profiles of 10 breast cancers from clinical tissue bank.</p> <p>Results</p> <p>Using hierarchical clustering analysis, 12 genes were found to be differentially expressed between the FNABs taken before and after surgical removal. Remarkably, most of the genes were linked to FOS in an early hypoxia pathway. The gene expression of FOS also increased with longer exposure to hypoxia.</p> <p>Conclusion</p> <p>Our study demonstrated that the timing of fine needle aspiration biopsies can be a confounding factor in microarray data analyses in breast cancer. We have shown that FOS-related genes, which have been implicated in early hypoxia as well as the development of breast cancers, were differentially expressed before and after surgery. Therefore, it is important that future studies take timing of tissue acquisition into account.</p

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically

Heme oxygenase-1 and carbon monoxide in pulmonary medicine

Heme oxygenase-1 (HO-1), an inducible stress protein, confers cytoprotection against oxidative stress in vitro and in vivo. In addition to its physiological role in heme degradation, HO-1 may influence a number of cellular processes, including growth, inflammation, and apoptosis. By virtue of anti-inflammatory effects, HO-1 limits tissue damage in response to proinflammatory stimuli and prevents allograft rejection after transplantation. The transcriptional upregulation of HO-1 responds to many agents, such as hypoxia, bacterial lipopolysaccharide, and reactive oxygen/nitrogen species. HO-1 and its constitutively expressed isozyme, heme oxygenase-2, catalyze the rate-limiting step in the conversion of heme to its metabolites, bilirubin IXα, ferrous iron, and carbon monoxide (CO). The mechanisms by which HO-1 provides protection most likely involve its enzymatic reaction products. Remarkably, administration of CO at low concentrations can substitute for HO-1 with respect to anti-inflammatory and anti-apoptotic effects, suggesting a role for CO as a key mediator of HO-1 function. Chronic, low-level, exogenous exposure to CO from cigarette smoking contributes to the importance of CO in pulmonary medicine. The implications of the HO-1/CO system in pulmonary diseases will be discussed in this review, with an emphasis on inflammatory states