Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.
Subject Categories Cardiovascular System; Metabolism
See also: C Kupatt and R Hinkel (March 2014)
angiogenesis; endothelial cell; ischemia; metabolism; VEGF-B
Small-molecule inhibition of hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) is being explored for the treatment of anemia. Previous studies have suggested that HIF-P4H-2 inhibition may also protect the heart from an ischemic insult. Hif-p4h-2gt/gt mice, which have 76 to 93% knockdown of Hif-p4h-2 mRNA in endothelial cells, fibroblasts, and cardiomyocytes and normoxic stabilization of Hif-α, were subjected to ligation of the left anterior descending coronary artery (LAD). Hif-p4h-2 deficiency resulted in increased survival, better-preserved left ventricle (LV) systolic function, and a smaller infarct size. Surprisingly, a significantly larger area of the LV remained perfused during LAD ligation in Hif-p4h-2gt/gt hearts than in wild-type hearts. However, no difference was observed in collateral vessels, while the size of capillaries, but not their number, was significantly greater in Hif-p4h-2gt/gt hearts than in wild-type hearts. Hif-p4h-2gt/gt mice showed increased cardiac expression of endothelial Hif target genes for Tie-2, apelin, APJ, and endothelial nitric oxide (NO) synthase (eNOS) and increased serum NO concentrations. Remarkably, blockage of Tie-2 signaling was sufficient to normalize cardiac apelin and APJ expression and resulted in reversal of the enlarged-capillary phenotype and ischemic cardioprotection in Hif-p4h-2gt/gt hearts. Activation of the hypoxia response by HIF-P4H-2 inhibition in endothelial cells appears to be a major determinant of ischemic cardioprotection and justifies the exploration of systemic small-molecule HIF-P4H-2 inhibitors for ischemic heart disease.
The endothelial Tie1 receptor is ligand-less, but interacts with the Tie2 receptor for angiopoietins (Angpt). Angpt2 is expressed in tumor blood vessels, and its blockade inhibits tumor angiogenesis. Here we found that Tie1 deletion from the endothelium of adult mice inhibits tumor angiogenesis and growth by decreasing endothelial cell survival in tumor vessels, without affecting normal vasculature. Treatment with VEGF or VEGFR-2 blocking antibodies similarly reduced tumor angiogenesis and growth; however, no additive inhibition was obtained by targeting both Tie1 and VEGF/VEGFR-2. In contrast, treatment of Tie1-deficient mice with a soluble form of the extracellular domain of Tie2, which blocks Angpt activity, resulted in additive inhibition of tumor growth. Notably, Tie1 deletion decreased sprouting angiogenesis and increased Notch pathway activity in the postnatal retinal vasculature, while pharmacological Notch suppression in the absence of Tie1 promoted retinal hypervasularization. Moreover, substantial additive inhibition of the retinal vascular front migration was observed when Angpt2 blocking antibodies were administered to Tie1-deficient pups. Thus, Tie1 regulates tumor angiogenesis, postnatal sprouting angiogenesis, and endothelial cell survival, which are controlled by VEGF, Angpt, and Notch signals. Our results suggest that targeting Tie1 in combination with Angpt/Tie2 has the potential to improve antiangiogenic therapy.
We report the first draft genome sequences of two Yersinia pseudotuberculosis sequence type 43 (ST43) (O:1b) strains, B-7194 and B-7195, isolated in Russia. The total lengths of the assemblies are 4,427,121 bp and 4,608,472 bp, and 3,819 and 4,018 coding sequences, respectively, were predicted within the genomes.
Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin-1 (ANG1)-mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1-dependent Phd2 repression initiated a feed-forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2-like phenotype, suggesting possible new venues for the treatment of ischaemic disorders.
arteriogenesis; ischaemia; macrophages; PHD2; TIE2
We report the first draft genome sequences of five Yersinia pseudotuberculosis isolates of sequence type (ST) 19 and of a variant from one of the five isolates. The total length of assemblies ranged from 4,226,485 bp to 4,274,148 bp, including between 3,808 and 3,843 predicted coding sequences.
Peroxisome proliferator-activated receptor (PPAR) delta is an important regulator of fatty acid (FA) metabolism. Angiopoietin-like 4 (Angptl4), a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL) activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR), PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.
φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.
Angiopoietin-2 (Ang2), a ligand for endothelial TEK (Tie2) tyrosine kinase receptor, is induced in hypoxic endothelial cells of tumors, where it promotes tumor angiogenesis and growth. However, the effects of Ang2 on tumor lymphangiogenesis and metastasis are poorly characterized.
We addressed the effect of Ang2 on tumor progression and metastasis using systemic Ang2 overexpression in mice carrying tumor xenografts, endothelium-specific overexpression of Ang2 in VEC-tTA/Tet-OS-Ang2 transgenic mice implanted with isogenic tumors, and administration of Ang2-blocking antibodies to tumor-bearing immunodeficient mice. Fisher's exact test was used for analysis of metastasis occurrence, and repeated measures one-way analysis of variance was used for the analysis of primary tumor growth curves. Unpaired t test was used for all other analyses. All statistical tests were two-sided.
Adenoviral expression of Ang2 increased lymph node and lung metastasis in tumor xenografts. The metastatic burden in the lungs was increased in transgenic mice in which Ang2 expression was induced specifically in the vascular endothelium (tumor burden per grid, VEC-tTA/Tet-OS-Ang2 mice [n = 5] vs control mice [n = 4]: 45.23 vs 12.26 mm2, difference = 32.67 mm2, 95% confidence interval = 31.87 to 34.07, P < .001). Ang2-blocking antibodies reduced lymph node and lung metastasis, as well as tumor lymphangiogenesis, and decreased tumor cell homing to the lungs after intravenous injection. In the lung metastases, Ang2 overexpression decreased endothelial integrity, whereas the Ang2-blocking antibodies improved endothelial cell–cell junctions and basement membrane contacts of metastasis-associated lung capillaries. At the cellular level, the Ang2-blocking antibodies induced the internalization of Ang2-Tie2 receptor complexes from endothelial cell–cell junctions in endothelial–tumor cell cocultures.
Our results indicate that blocking Ang2 inhibits metastatic dissemination in part by enhancing the integrity of endothelial cell–cell junctions.
The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium and is generally conserved between the epidemic strains of Yersinia pestis. We investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from 19 Y. pestis strains belonging to different phylogenetic groups (“subspecies”) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324–326. These major variations, together with other minor substitutions in amino acid sequences, allowed us to classify the LcrV alleles into five sequence types (A-E). We observed that the strains of different Y. pestis “subspecies” can have the same type of LcrV, including that conserved in epidemic strains, and different types of LcrV can exist within the same natural plague focus. Therefore, the phenomenon of “selective virulence” characteristic of the strains of the microtus biovar is unlikely to be the result of polymorphism of the V antigen. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in the oligomerization of LcrV.
LcrV; Yersinia pestis; polymorphism; 3D structure; modeling; oligomerization
The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane β-barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37°C than in cells grown at 20°C; the difference in enzymatic activity by far exceeded the increase in the cellular content of the Pla protein. Y. pestis modifies its LPS structure in response to growth temperature. We purified His6-Pla under denaturing conditions and compared various LPS types for their capacity to enhance plasmin formation by His6-Pla solubilized in detergent. Reactivation of His6-Pla was higher with Y. pestis LPSs isolated from bacteria grown at 37°C than with LPSs from cells grown at 25°C. Lack of O antigens and the presence of the outer core region as well as a lowered level of acylation in LPS were found to enhance the Pla-LPS interaction. Genetic substitution of arginine 138, which is part of a three-dimensional protein motif for binding to lipid A phosphates, decreased both the enzymatic activity of His6-Pla and the amount of Pla in Y. pestis cells, suggesting the importance of the Pla-lipid A phosphate interaction. The temperature-induced changes in LPS are known to help Y. pestis to avoid innate immune responses, and our results strongly suggest that they also potentiate Pla-mediated proteolysis.
The therapeutic potential of vascular endothelial growth factor C (VEGF-C) and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly employed adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the four-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system.
VEGF-C; VEGF-D; adeno-associated virus; angiogenesis; lymphangiogenesis; skeletal muscle
The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/-3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/-3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.
angiogenic sprouting; embryoid body; heterodimer; proximity ligation; VEGF receptor
The species Yersinia pestis is commonly divided into three classical biovars, Antiqua, Medievalis, and Orientalis, belonging to subspecies pestis pathogenic for human and the (atypical) non-human pathogenic biovar Microtus (alias Pestoides) including several non-pestis subspecies. Recent progress in molecular typing methods enables large-scale investigations in the population structure of this species. It is now possible to test hypotheses about its evolution which were proposed decades ago. For instance the three classical biovars of different geographical distributions were suggested to originate from Central Asia. Most investigations so far have focused on the typical pestis subspecies representatives found outside of China, whereas the understanding of the emergence of this human pathogen requires the investigation of strains belonging to subspecies pestis from China and to the Microtus biovar.
Multi-locus VNTR analysis (MLVA) with 25 loci was performed on a collection of Y. pestis isolates originating from the majority of the known foci worldwide and including typical rhamnose-negative subspecies pestis as well as rhamnose-positive subspecies pestis and biovar Microtus. More than 500 isolates from China, the Former Soviet Union (FSU), Mongolia and a number of other foci around the world were characterized and resolved into 350 different genotypes. The data revealed very close relationships existing between some isolates from widely separated foci as well as very high diversity which can conversely be observed between nearby foci.
The results obtained are in full agreement with the view that the Y. pestis subsp. pestis pathogenic for humans emerged in the Central Asia region between China, Kazakhstan, Russia and Mongolia, only three clones of which spread out of Central Asia. The relationships among the strains in China, Central Asia and the rest of the world based on the MLVA25 assay provide an unprecedented view on the expansion and microevolution of Y. pestis.
Yersinia pestis, the pathogen of plague, has greatly influenced human history on a global scale. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), an element participating in immunity against phages' invasion, is composed of short repeated sequences separated by unique spacers and provides the basis of the spoligotyping technology. In the present research, three CRISPR loci were analyzed in 125 strains of Y. pestis from 26 natural plague foci of China, the former Soviet Union and Mongolia were analyzed, for validating CRISPR-based genotyping method and better understanding adaptive microevolution of Y. pestis.
Using PCR amplification, sequencing and online data processing, a high degree of genetic diversity was revealed in all three CRISPR elements. The distribution of spacers and their arrays in Y. pestis strains is strongly region and focus-specific, allowing the construction of a hypothetic evolutionary model of Y. pestis. This model suggests transmission route of microtus strains that encircled Takla Makan Desert and ZhunGer Basin. Starting from Tadjikistan, one branch passed through the Kunlun Mountains, and moved to the Qinghai-Tibet Plateau. Another branch went north via the Pamirs Plateau, the Tianshan Mountains, the Altai Mountains and the Inner Mongolian Plateau. Other Y. pestis lineages might be originated from certain areas along those routes.
CRISPR can provide important information for genotyping and evolutionary research of bacteria, which will help to trace the source of outbreaks. The resulting data will make possible the development of very low cost and high-resolution assays for the systematic typing of any new isolate.
Lipopolysaccharide (LPS) structure impacts the bactericidal action of cationic peptides, such as polymyxin B (PMB), and sensitivity to killing by normal human serum (NHS). Cultivation of different subspecies strains of Yersinia pestis isolated from unrelated geographic origins at various temperatures (mammals, 37°C; fleas, 25°C; or winter hibernation, 6°C) affects LPS composition and structure. We tested the susceptibilities of various strains of Y. pestis grown at these different temperatures to PMB and serum bactericidal killing. Both properties varied significantly in response to temperature changes. In Y. pestis subsp. pestis (the main subspecies causing human plague), high levels of resistance to PMB and NHS were detected at 25°C. However, at the same temperature, Y. pestis subsp. caucasica was highly sensitive to PMB. At both of the extreme temperatures, all strains were highly susceptible to PMB. At 25°C and 37°C, Y. pestis subsp. caucasica strain 1146 was highly susceptible to the bactericidal activity of 80% NHS. All Y. pestis strains studied were able to grow in heat-inactivated human serum or in 80% normal mouse serum. At 6°C, all strains were highly sensitive to NHS. Variations in the PMB resistance of different bacterial cultures related to both the content of cationic components (4-amino-4-deoxyarabinose in lipid A and glycine in the core) and a proper combination of terminal monosaccharides in the LPS. The NHS resistance correlated with an elevated content of N-acetylglucosamine in the LPS. Structural variation in the LPS of Y. pestis correlates with the organism's ability to resist innate immunity in both fleas and mammals.
Increased interest in the pathogenic potential of Yersinia pestis has emerged because of the potential threats from bioterrorism. Pathogenic potential is based on genetic factors present in a population of microbes, yet most studies evaluating the role of specific genes in virulence have used a limited number of strains. For Y. pestis this issue is complicated by the fact that most strains available for study in the Americas are clonally derived and thus genetically restricted, emanating from a strain of Y. pestis introduced into the United States in 1902 via marine shipping and subsequent spread of this strain throughout North and South America. In countries from the former Soviet Union (FSU), Mongolia, and China there are large areas of enzootic foci of Y. pestis infection containing genetically diverse strains that have been intensely studied by scientists in these countries. However, the results of these investigations are not generally known outside of these countries. Here we describe the variety of methods used in the FSU to classify Y. pestis strains based on genetic and phenotypic variation and show that there is a high level of diversity in these strains not reflected by ones obtained from sylvatic areas and patients in the Americas.