Cancer and stromal cells actively exert physical forces (solid stress) to compress tumour blood vessels, thus reducing vascular perfusion. Tumour interstitial matrix also contributes to solid stress, with hyaluronan implicated as the primary matrix molecule responsible for vessel compression because of its swelling behaviour. Here we show, unexpectedly, that hyaluronan compresses vessels only in collagen-rich tumours, suggesting that collagen and hyaluronan together are critical targets for decompressing tumour vessels. We demonstrate that the angiotensin inhibitor losartan reduces stromal collagen and hyaluronan production, associated with decreased expression of profibrotic signals TGF-β1, CCN2 and ET-1, downstream of angiotensin-II-receptor-1 inhibition. Consequently, losartan reduces solid stress in tumours resulting in increased vascular perfusion. Through this physical mechanism, losartan improves drug and oxygen delivery to tumours, thereby potentiating chemotherapy and reducing hypoxia in breast and pancreatic cancer models. Thus, angiotensin inhibitors —inexpensive drugs with decades of safe use — could be rapidly repurposed as cancer therapeutics.

Purpose Recent clinical trials of antivascular endothelial growth factor (VEGF) agents for glioblastoma showed promising progression-free and overall survival rates. However, available clinical imaging does not separate antitumor effects from antipermeability effects of these agents. Thus although anti-VEGF agents may decrease tumor contrast-enhancement, vascularity, and edema, the mechanisms leading to improved survival in patients remain incompletely understood. Our goal was to determine whether alleviation of edema by anti-VEGF agents alone could increase survival in mice. Methods We treated mice bearing three different orthotopic models of glioblastoma with a VEGF-targeted kinase inhibitor, cediranib. Using intravital microscopy, molecular techniques, and magnetic resonance imaging (MRI), we measured survival, tumor growth, edema, vascular morphology and function, cancer cell apoptosis and proliferation, and circulating angiogenic biomarkers. Results We show by intravital microscopy that cediranib significantly decreased tumor vessel permeability and diameter. Moreover, cediranib treatment induced normalization of perivascular cell coverage and thinning of the basement membrane, as mirrored by an increase in plasma collagen IV. These rapid changes in tumor vascular morphology and function led to edema alleviation—as measured by MRI and by dry/wet weight measurement of water content—but did not affect tumor growth. By immunohistochemistry, we found a transient decrease in macrophage infiltration and significant but minor changes in tumor cell proliferation and apoptosis. Systemically, cediranib increased plasma VEGF and placenta growth factor levels, and the number of circulating CXCR4+CD45+ cells. However, by controlling edema, cediranib significantly increased survival of mice in the face of persistent tumor growth. Conclusion Anti-VEGF agents may be able to improve survival of patients with glioblastoma, even without inhibiting tumor growth.

Not all tumor vessels are equal. Tumor-associated vasculature includes immature vessels, regressing vessels, transport vessels undergoing arteriogenesis and peritumor vessels influenced by tumor growth factors. Current techniques for analyzing tumor blood flow do not discriminate between vessel subtypes and only measure average changes from a population of dissimilar vessels. We have developed methodologies for simultaneously quantifying blood flow (velocity, flux, hematocrit and shear rate) in extended networks at single capillary resolution in vivo. Our approach relies on deconvolution of signals produced by labeled red blood cells as they move relative to the scanning laser of a confocal or multiphoton microscope and provides fully-resolved three-dimensional flow profiles within vessel networks. Using this methodology, we show that blood velocity profiles are asymmetric near intussusceptive tissue structures in tumors in mice. Furthermore, we show that subpopulations of vessels, classified by functional parameters, exist in, around a tumor and in normal brain.

Rapid blood perfusion is critical for postimplantation survival of thick, prevascularized bioartificial tissues. Yet the mechanism by which implanted vascular networks inosculate, or anastomose, with the host vasculature has been unknown, making it difficult to develop optimized strategies for facilitating perfusion. Here we show that implanted vascular networks anastomose with host vessels through a previously unidentified process of "wrapping and tapping" between the engrafted endothelial cells (ECs) and the host vasculature. At the host-implant interface, implanted ECs first wrap around nearby host vessels and then cause basement membrane and pericyte reorganization and localized displacement of the underlying host endothelium. In this way, the implanted ECs replace segments of host vessels to divert blood flow to the developing implanted vascular network. IntroductionA major obstacle in tissue engineering is poor postimplantation graft survival because of insufficient blood perfusion. A potential solution is to populate the engineered tissue with endothelial cells (ECs) or endothelial progenitor cells (EPCs), which can quickly organize into interconnected networks, undergo lumenogenesis, and anastomose with the host vasculature to redirect blood flow into the graft. 1 Human umbilical vein endothelial cells (HUVECs), [2][3][4] ECs derived from human embryonic stem cells, 5 and human adult and cord blood EPCs 6,7 are all capable of generating such patent vascular networks in vivo. This approach has proven effective in improving the quality of engineered skeletal muscle 8 and bone 9 tissues.Limited studies suggest that, during embryonic vasculogenesis and sprouting angiogenesis, anastomosis is accomplished via connection of extended cellular processes followed by lumen propagation through intracellular and intercellular vacuole fusion, 10,11 with macrophages playing an accessory role. 12 However, it is not known whether this is the only mechanism for connecting vessels. Without a basic understanding of the cellular mechanisms of anastomosis, it is difficult to develop strategies for accelerating this critical step for perfusing engrafted tissues.To investigate the process of anastomosis, we used a previously established model in which HUVECs and mouse mesenchymal precursor cells are embedded in collagen-fibronectin gels and placed in cranial window preparations of severe combined immunodeficient mice 2 (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). In this system, anastomosis between host vessels and implanted EC networks occurs as early as 2 weeks after implantation, and the engineered vessels remain stable and functional for one year in vivo. 2 Similar results can be achieved when the mouse mesenchymal precursor cells are replaced with human mesenchymal stem cells 13 or human lung fibroblasts. 14,15 Tracking fluorescently labeled implanted ECs and host ECs simultaneously in live animals, we found that tip cell connections and va...

A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021. Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain. This phenotype is attributed to reduction in succinoglycan synthesis. We have used complementation of this phenotype and the previously described D-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene. Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B. japonicum genome. The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin.

e Lactic acid bacteria are found in the gastrointestinal tract of mammals and have received tremendous attention due to their health-promoting properties. We report the development of two dual-color luciferase-producing Lactobacillus (Lb.) plantarum and Lactococcus (Lc.) lactis strains for noninvasive simultaneous tracking in the mouse gastrointestinal tract. We previously described the functional expression of the red luciferase mutant (CBRluc) from Pyrophorus plagiophthalamus in Lb. plantarum NCIMB8826 and Lc. lactis MG1363 (C. Daniel, S. Poiret, V. Dennin, D. Boutillier, and B. Pot, Appl Environ Microbiol 79:1086 -1094, 2013, http://dx.doi.org/10.1128/AEM.03221-12). In this study, we determined that CBRluc is a better-performing luciferase for in vivo localization of both lactic acid bacteria after oral administration than the green click beetle luciferase mutant construct developed in this study. We further established the possibility to simultaneously detect red-and green-emitting lactic acid bacteria by dual-wavelength bioluminescence imaging in combination with spectral unmixing. The difference in spectra of light emission by the red and green click beetle luciferase mutants and dual bioluminescence detection allowed in vitro and in vivo quantification of the red and green emitted signals; thus, it allowed us to monitor the dynamics and fate of the two bacterial populations simultaneously. Persistence and viability of both strains simultaneously administered to mice in different ratios was studied in vivo in anesthetized mice and ex vivo in mouse feces. The application of dual-luciferase-labeled bacteria has considerable potential to simultaneously study the interactions and potential competitions of different targeted bacteria and their hosts. L actococci and lactobacilli are lactic acid bacteria (LAB) that have been used for thousands of years for the production and preservation of fermented food, such as milk, vegetables, and meat. Some specific strains are commercialized as probiotics and claimed to have health-promoting properties (1). The gastrointestinal tract (GIT) is the most important field of activity of LAB, although distal effects outside the gut have been described (2). Thus, it is important to understand the interactions of the administered bacteria with their host GIT system. LAB are able to survive and adapt to the GIT conditions, as shown by comparative and functional genomic characterization of various human isolates, essentially lactobacilli (for a review, see references 2 and 3). Some LAB, when present in the GIT of mice or humans, express a number of common characteristics that may relate to their intestinal niche adaptation (3, 4). However, direct in vivo tracking of these actions in terms of both spatial and temporal evolution would allow a better understanding of the survival and metabolic activities of these LAB in the gut.In the past decade, bioluminescence imaging (BLI) has become essential for in vivo noninvasive monitoring of biological processes (4). The technique relies on ...

Blood flow directly affects nutrient and oxygen as well drug delivery in tumors. The distribution of blood within tumor is disturbed by structural and functional abnormalities, and may be further altered by therapy. In turn, this results in heterogeneous distribution of drugs and/or oxygen, with direct effects on the efficacy of cytotoxic therapies. These heterogeneities are poorly understood because of the inability of current techniques to characterize the function of individual vessels and their local network. To address this problem, we have developed a novel methodology for simultaneously quantifying blood flow (velocity, flux, hematocrit and shear rate) in extended networks at the single capillary level. Our approach relies on deconvolution of signals produced by labeled red blood cells as they move relative to the scanning laser of a confocal or multiphoton microscope, and provides fully‐resolved three‐dimensional flow profiles within vessel networks. Using this methodology, we show that blood velocity profiles are perturbed and asymmetrical near intussusceptive tissue structures in tumors. Furthermore, we show that subpopulations of vessels, classified by functional parameters, exist in different tissue regions, and that Angiopeitin‐1 can improve tumor perfusion by inducing arteriogenesis.

A major obstacle in tissue engineering is poor post‐implantation cell survival due to the lack of blood perfusion. One approach is to populate the graft before implantation with endothelial or progenitor cells that can quickly organize into a network and connect with the host vasculature to achieve blood perfusion. Many types of endothelial and endothelial progenitor cells are capable of this, but the mechanism by which the implanted vascular networks anastomose with the host vasculature is unknown. Here we show that it occurs through a previously unreported process of “wrapping and tapping” (WAT). At the host‐implant interface, implanted ECs wrap around nearby host vessels, cause host vessel regression by disrupting their pericytes and basement membrane, and then completely replace segments of host vessels to divert flow into the contiguous, engrafted vascular network. This process is facilitated by high levels of MMP‐14 and MMP‐9 expressed specifically by wrapping ECs. Our results provide mechanistic understanding on this critical step of vascularization and may have implications for other physiological and pathological processes involving post‐natal vasculogenesis.