Kishor K. Sivaraj

Platelet-Derived Nucleotides Promote Tumor-Cell Transendothelial Migration and Metastasis via P2Y2 Receptor

Tumor cells can activate platelets, which in turn facilitate tumor cell survival and dissemination. The exact mechanisms by which platelets promote metastasis have remained unclear. Here, we show that adenine nucleotides released from tumor cell-activated platelets induce opening of the endothelial barrier to allow transendothelial migration of tumor cells and thereby promote cancer cell extravasation. We identified the endothelial P2Y2 receptor, which is activated by ATP, as the primary mediator of this effect. Mice deficient in P2Y2 or lacking ATP secretion from platelets show strongly reduced tumor cell metastasis. These findings demonstrate a mechanism by which platelets promote cancer cell metastasis and suggest the P2Y2 receptor and its endothelial downstream signaling mechanisms as a target for antimetastatic therapies.

ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis

Diagnosis of metastatic breast cancer is associated with a very poor prognosis. New therapeutic targets are urgently needed, but their development is hampered by a lack of understanding of the mechanisms leading to tumor metastasis. Exemplifying this is the fact that the approximately 30% of all breast cancers overexpressing the receptor tyrosine kinase ErbB-2 are characterized by high metastatic potential and poor prognosis, but the signaling events downstream of ErbB-2 that drive cancer cell invasion and metastasis remain incompletely understood. Here we show that overexpression of ErbB-2 in human breast cancer cell lines leads to phosphorylation and activation of the semaphorin receptor Plexin-B1. This was required for ErbB-2-dependent activation of the pro-metastatic small GTPases RhoA and RhoC and promoted invasive behavior of human breast cancer cells. In a mouse model of ErbB-2-overexpressing breast cancer, ablation of the gene encoding Plexin-B1 strongly reduced the occurrence of metastases. Moreover, in human patients with ErbB-2-overexpressing breast cancer, low levels of Plexin-B1 expression correlated with good prognosis. Our data suggest that Plexin-B1 represents a new candidate therapeutic target for treating patients with ErbB-2-positive breast cancer.

Blood vessel formation and function in bone

In addition to their conventional role as a conduit system for gases, nutrients, waste products or cells, blood vessels in the skeletal system play active roles in controlling multiple aspects of bone formation and provide niches for hematopoietic stem cells that reside within the bone marrow. In addition, recent studies have highlighted roles for blood vessels during bone healing. Here, we provide an overview of the architecture of the bone vasculature and discuss how blood vessels form within bone, how their formation is modulated, and how they function during development and fracture repair. Summary: This Review discusses the roles of blood vessels in controlling developmental and regenerative bone formation and providing niche microenvironments for various cell types.

G13 Controls Angiogenesis through Regulation of VEGFR-2 Expression

At sites of angiogenesis, the expression of the key angiogenesis regulator vascular endothelial growth factor (VEGF) and its main receptor, VEGF receptor 2 (VEGFR-2), are strongly upregulated. Whereas the processes controlling VEGF expression are well described, the mechanisms underlying VEGFR-2 upregulation have remained unclear. We found that endothelial VEGFR-2 expression is strongly reduced in the absence of the G protein G13, resulting in an impaired responsiveness to VEGF-A, a phenotype that can be rescued by normalization of VEGFR-2 levels. G13-mediated VEGFR-2 expression involved activation of the small GTPase RhoA and transcription factor NF-κB, the latter acting via a specific binding site at position -84 of the VEGFR-2 promoter. Mice with endothelial cell-specific loss of G13 showed reduced VEGFR-2 expression at sites of angiogenesis and attenuated VEGF effects, resulting in impaired retinal angiogenesis and tumor vascularization. Taken together, we identified G-protein-mediated signaling via G13 as a critical regulator of VEGFR-2 expression during angiogenesis.

Cell-matrix signals specify bone endothelial cells during developmental osteogenesis

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell–matrix signalling interactions. Loss of endothelial integrin β1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.

Flow Dynamics and HSPC Homing in Bone Marrow Microvessels

Summary Measurements of flow velocities at the level of individual arterial vessels and sinusoidal capillaries are crucial for understanding the dynamics of hematopoietic stem and progenitor cell homing in the bone marrow vasculature. We have developed two complementary intravital two-photon imaging approaches to determine blood flow dynamics and velocities in multiple vessel segments by capturing the motion of red blood cells. High-resolution spatiotemporal measurements through a cranial window to determine short-time dynamics of flowing blood cells and repetitive centerline scans were used to obtain a detailed flow-profile map with hemodynamic parameters. In addition, we observed the homing of individual hematopoietic stem and progenitor cells and obtained detailed information on their homing behavior. With our imaging setup, we determined flow patterns at cellular resolution, blood flow velocities and wall shear stress in small arterial vessels and highly branched sinusoidal capillaries, and the cellular dynamics of hematopoietic stem and progenitor cell homing.

Endothelial Gαq/11 is required for VEGF-induced vascular permeability and angiogenesis

AIMS VEGF A (VEGF-A) is a central regulator of pre- and postnatal vascular development. In vitro studies suggested that heterotrimeric G-proteins of the Gq/11 family contribute to VEGF receptor 2 (VEGFR2) signalling, but the mechanism and physiological relevance of this finding is unknown. The aim of this study is to understand the role of endothelial Gαq/11 in VEGF-dependent regulation of vascular permeability and angiogenesis. METHODS AND RESULTS We show here that VEGF-A-induced signalling events, such as VEGFR2 autophosphorylation, calcium mobilization, or phosphorylation of Src and Cdh5, were reduced in Gαq/11-deficient endothelial cells (ECs), resulting in impaired VEGF-dependent barrier opening, tube formation, and proliferation. Agonists at Gq/11-coupled receptors facilitated VEGF-A-induced VEGFR2 autophosphorylation in a Gαq/11-dependent manner, thereby enhancing downstream VEGFR2 signalling. In vivo, EC-specific Gαq/11- and Gαq-deficient mice showed reduced VEGF-induced fluid extravasation, and retinal angiogenesis was significantly impaired. Gαq-deficient ECs showed reduced proliferation, Cdh5 phosphorylation, and fluid extravasation, whereas apoptosis was increased. CONCLUSION Gαq/11 critically contributes to VEGF-A-dependent permeability control and angiogenic behaviour in vitro and in vivo.

S1P2/G12/13 Signaling Negatively Regulates Macrophage Activation and Indirectly Shapes the Atheroprotective B1-Cell Population

Objectives— Monocyte/macrophage recruitment and activation at vascular predilection sites plays a central role in the pathogenesis of atherosclerosis. Heterotrimeric G proteins of the G12/13 family have been implicated in the control of migration and inflammatory gene expression, but their function in myeloid cells, especially during atherogenesis, is unknown. Approach and Results— Mice with myeloid-specific deficiency for G12/13 show reduced atherosclerosis with a clear shift to anti-inflammatory gene expression in aortal macrophages. These changes are because of neither altered monocyte/macrophage migration nor reduced activation of inflammatory gene expression; on the contrary, G12/13-deficient macrophages show an increased nuclear factor-&kgr;B–dependent gene expression in the resting state. Chronically increased inflammatory gene expression in resident peritoneal macrophages results in myeloid-specific G12/13-deficient mice in an altered peritoneal micromilieu with secondary expansion of peritoneal B1 cells. Titers of B1-derived atheroprotective antibodies are increased, and adoptive transfer of peritoneal cells from mutant mice conveys atheroprotection to wild-type mice. With respect to the mechanism of G12/13-mediated transcriptional control, we identify an autocrine feedback loop that suppresses nuclear factor-&kgr;B–dependent gene expression through a signaling cascade involving sphingosine 1-phosphate receptor subtype 2, G12/13, and RhoA. Conclusions— Together, these data show that selective inhibition of G12/13 signaling in macrophages can augment atheroprotective B-cell populations and ameliorate atherosclerosis.

Response to Harper et al.

We thank Harper et al. for their comments on our publication. The authors of this correspondence point to some differences between our publication (Schumacher et al., 2013xSchumacher, D., Strilic, B., Sivaraj, K.K., Wettschureck, N., and Offermanns, S. Cancer Cell. 2013; 24: 130–137Abstract | Full Text | Full Text PDF | PubMed | Scopus (77)See all ReferencesSchumacher et al., 2013) and others with regard to the in vitro phenotype of Munc13-4-deficient platelets. Based on our experimental data, we think that Munc13-4 deficiency primarily blocks the release from dense granules, but not from α-granules of platelets. It is, however, well known that platelet activation involves multiple positive feedback loops and that the release of ATP/ADP from dense granules can promote platelet aggregation and α-granule release via the autocrine and paracrine activation of P2Y1 and P2Y12 receptors (Gachet, 2006xGachet, C. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 277–300Crossref | PubMed | Scopus (195)See all References, Kahner et al., 2006xKahner, B.N., Shankar, H., Murugappan, S., Prasad, G.L., and Kunapuli, S.P. J. Thromb. Haemost. 2006; 4: 2317–2326Crossref | PubMed | Scopus (126)See all References). Based on this concept, it is indeed expected that loss of ATP/ADP release from platelet dense granules also affects, under certain experimental conditions, the release of α-granules from activated platelets, as well as the aggregation of platelets. Whether these secondary effects of a blocked dense-granule release are seen in vitro depends on the experimental conditions used to study platelet function, such as platelet concentration or the concentration and type of applied stimuli. At intermediate thrombin concentrations, we actually saw a tendency of reduced PF4 release and integrin αIIbβ3 activation which, however, was not significant (Figures 2A and S2C in Schumacher et al., 2013xSchumacher, D., Strilic, B., Sivaraj, K.K., Wettschureck, N., and Offermanns, S. Cancer Cell. 2013; 24: 130–137Abstract | Full Text | Full Text PDF | PubMed | Scopus (77)See all ReferencesSchumacher et al., 2013). In the experiments mentioned by Harper et al., we used relatively low platelet concentrations, reducing the likelihood that sufficient amounts of ATP/ADP are released to induce secondary effects. Finally, at least in the case of tumor-cell-induced platelet activation, it is also possible that the small amount of ATP released from tumor cells (see Figure 1D in Schumacher et al., 2013xSchumacher, D., Strilic, B., Sivaraj, K.K., Wettschureck, N., and Offermanns, S. Cancer Cell. 2013; 24: 130–137Abstract | Full Text | Full Text PDF | PubMed | Scopus (77)See all ReferencesSchumacher et al., 2013) obscured a somewhat reduced α-granule release.Whether the secondary effects of dense-granule release are of relevance for platelet-dependent tumor cell metastasis is not known. Nevertheless, we agree that solely on the basis of our experiments performed with Munc13-4-deficient platelets and animals, we would not be able to exclude that defects other than reduced dense granule secretion also contribute to the observed defects in transendothelial tumor cell migration and in vivo metastasis. We therefore mentioned in the discussion of this publication the possibility that “in addition to ATP, other platelet-derived factors, such as transforming growth factor β, promote, in particular, later stages of tumor cell extravasation (Labelle et al., 2011xLabelle, M., Begum, S., and Hynes, R.O. Cancer Cell. 2011; 20: 576–590Abstract | Full Text | Full Text PDF | PubMed | Scopus (299)See all ReferencesLabelle et al., 2011).”However, our study goes beyond the description of the role of dense-granule secretion in platelet-dependent tumor cell transendothelial migration and metastasis. We also identified a downstream mechanism by demonstrating that the endothelial ATP receptor P2Y2 mediates platelet-dependent tumor cell transmigration and that loss of P2Y2, which is not present on platelets, strongly reduces tumor cell metastasis from primary tumors, as well as after intravenous injection of tumor cells. If the above described secondary effects played a major role in platelet dense-granule secretion-dependent tumor cell transendothelial migration and metastasis, we would expect the phenotype of Munc13-4 deficiency to be more severe than the phenotype of P2Y2 deficiency. However, both platelet Munc13-4 deficiency and endothelial knockdown of P2Y2 abolished platelet-dependent transendothelial tumor cell migration. More importantly, tumor cell metastasis in vivo, when studied in bone-marrow chimeras, was reduced both in P2Y2-deficient mice transplanted with wild-type bone-marrow and in P2Y2-deficient mice transplanted with Munc13-4-deficient bone marrow to the same degree, and no additive effect could be seen.Thus, while we acknowledge that loss of dense-granule secretion in Munc13-4-deficient platelets can lead under certain experimental conditions to a secondary impairment of α-granule release, we think that our data show that dense-granule-derived ATP acting on P2Y2 receptors is the primary mechanism through which blockade of ATP release from Munc13-4-deficient platelets affects tumor cell transendothelial migration and metastasis.