Real-time Imaging of Myeloid Cells Dynamics in <em>Apc<sup>Min/+</sup></em> Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

University of California

The lymphatic vasculature is essential for the maintenance of normal fluid balance and for the immune responses, but it is also involved in a variety of diseases. Hypoplasia or dysfunction of the lymphatic vessels can lead to lymphedema, whereas hyperplasia or abnormal growth of these vessels are associated with lymphangiomas and lymphangiosarcomas. Lymphatic vessels are also involved in lymph node and systemic metastasis of cancer cells. Recent novel findings on the molecular mechanisms involved in lymphatic vessel development and regulation allow the modulation of the lymphangiogenic process and specific targeting of the lymphatic endothelium. Recent results show that the homeodomain transcription factor Prox-1 is an important lymphatic endothelial cell (LEC) fate-determining factor which can induce LEC-specific gene transcription even in blood vascular endothelial cells (BECs). This suggests that the distinct phenotypes of cells in the adult vascular endothelium are plastic and sensitive to transcriptional reprogramming, which might be useful for future therapeutic applications involving endothelial cells. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are peptide growth factors capable of inducing the growth of new lymphatic vessels in vivo in a process called lymphangiogenesis. They belong to the larger family which also includes VEGF, placenta growth factor (PlGF) and VEGF-B, VEGF-C and VEGF-D are ligands for the endothelial cell specific tyrosine kinase receptors VEGFR-2 and VEGFR-3. In adult human as well as mouse tissues VEGFR-3 is expressed predominantly in lymphatic endothelial cells which line the inner surface of lymphatic vessels. While VEGFR-2 is thought to be the main mediator of angiogenesis, VEGFR-3 signaling is crucial for the development of the lymphatic vessels. Heterozygous inactivation of the VEGFR-3 tyrosine kinase leads to primary lymphedema due to defective lymphatic drainage in the limbs. Other factors that seem to be involved in lymphangiogenesis include the Tie/angiopoietin system, neuropilin-2 and integrin alpha 9. VEGF-C induces lymphatic vessel growth, but high levels of VEGF-C also resulted in blood vessel leakiness and growth. The VEGFR-3-specific mutant form of VEGF-C called VEGF-C156S lacks blood vascular side effects but is sufficient for therapeutic lymphangiogenesis in a mouse model of lymphedema. As VEGF-C156S is a specific lymphatic endothelial growth factor in the skin, it provides an attractive molecule for pro-lymphangiogenic therapy.

Lymphangiogenic growth factors, receptors and therapies.

Vascular endothelial cells are characterized by a high degree of functional and phenotypic plasticity, which is controlled both by their pericellular microenvironment and their intracellular gene expression programs. To gain further insight into the mechanisms regulating the endothelial cell phenotype, we have compared the responses of lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) to vascular endothelial growth factors (VEGFs). VEGFR-3-specific signals are sufficient for LEC but not BEC proliferation, as shown by the ability of the specific ligand VEGF-C156S to stimulate cell cycle entry only in LECs. On the other hand, we found that VEGFR-3 stimulation did not induce LEC cell shape changes typical of VEGFR-2-stimulated LECs, indicating receptor-specific differences in the cytoskeletal responses. Genes induced via VEGFR-2 also differed between BECs and LECs: angiopoietin-2 (Ang-2) was induced via VEGFR-2 in BECs and LECs, but the smooth muscle cell (SMC) chemoattractant BMP-2 was induced only in BECs. Both BECs and LECs were able to promote SMC chemotaxis, but contact with SMCs led to down-regulation of VEGFR-3 expression in BECs in a 3-dimensional coculture system. This was consistent with the finding that VEGFR-3 is down-regulated in vivo at sites of endothelial cell-pericyte/smooth muscle cell contacts. Collectively, these data show intrinsic cell-specific differences of BEC and LEC responses to VEGFs and identify a pericellular regulatory mechanism for VEGFR-3 down-regulation in endothelial cells.

Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function.

Platelet-derived growth factor-D (PDGF-D) is a recently characterized member of the PDGF family with unknown in vivo functions. We investigated the effects of PDGF-D in transgenic mice by expressing it in basal epidermal cells and then analyzed skin histology, interstitial fluid pressure, and wound healing. When compared with control mice, PDGF-D transgenic mice displayed increased numbers of macrophages and elevated interstitial fluid pressure in the dermis. Wound healing in the transgenic mice was characterized by increased cell density and enhanced recruitment of macrophages. Macrophage recruitment was also the characteristic response when PDGF-D was expressed in skeletal muscle or ear by an adeno-associated virus vector. Combined expression of PDGF-D with vascular endothelial growth factor-E (VEGF-E) led to increased pericyte/smooth muscle cell coating of the VEGF-E-induced vessels and inhibition of the vascular leakiness that accompanies VEGF-E-induced angiogenesis. These results show that full-length PDGF-D is activated in tissues and is capable of increasing interstitial fluid pressure and macrophage recruitment and the maturation of blood vessels in angiogenic processes.

PDGF-D induces macrophage recruitment, increased interstitial pressure, and blood vessel maturation during angiogenesis.

Angiopoietin 1 (Ang1), a ligand for the receptor tyrosine kinase Tie2, regulates the formation and stabilization of the blood vessel network during embryogenesis. In adults, Ang1 is associated with blood vessel stabilization and recruitment of perivascular cells, whereas Ang2 acts to counter these actions. Recent results from gene-targeted mice have shown that Ang2 is also essential for the proper patterning of lymphatic vessels and that Ang1 can be substituted for this function. In order to characterize the effects of the angiopoietins on lymphatic vessels, we employed viral vectors for overexpression of Ang1 in adult mouse tissues. We found that Ang1 activated lymphatic vessel endothelial proliferation, vessel enlargement, and generation of long endothelial cell filopodia that eventually fused, leading to new sprouts and vessel development. Cutaneous lymphatic hyperplasia was also detected in transgenic mice expressing Ang1 in the basal epidermal cells. Tie2 was expressed in the lymphatic endothelial cells and Ang1 stimulation of these cells resulted in up-regulation of vascular endothelial growth factor receptor 3 (VEGFR-3). Furthermore, a soluble form of VEGFR-3 inhibited the observed lymphatic sprouting. Our results reinforce the concept that Ang1 therapy may be useful in settings of tissue edema.

Angiopoietin-1 promotes lymphatic sprouting and hyperplasia.

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.

Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in mouse embryos but leads to persistent lymphatic hyperplasia in adult tissues.

Vascular endothelial growth factors and their endothelial tyrosine kinase receptors are central regulators of vasculogenesis, angiogenesis and lymphangiogenesis. VEGF signalling through VEGFR-2 is the major angiogenic pathway, and blockage of VEGF/VEGFR-2 signalling is the first anti-angiogenic strategy for cancer therapy. VEGFR-1 seems to act as a negative regulator of VEGF-mediated angiogenesis during development, and as a stimulator of pathological angiogenesis when activated by its specific ligands PlGF and VEGF-B. PlGF recruits angiogenic macrophages to tumours, and targeting PlGF could therefore be beneficial in cancer. For VEGF-B, with very limited angiogenic potential, a new role has been identified in regulating lipid metabolism in the heart. VEGF-C and VEGF-D induce lymphangiogenesis via VEGFR-3 and have also been shown to be lymphangiogenic in tumours, stimulating metastasis. Mouse models of lymphoedema have established VEGF-C as a promising agent for pro-lymphangiogenic therapy. In addition to lymphangiogenesis, VEGFR-3 has also been shown to be important for angiogenesis, acting together with VEGF/VEGFR-2 and Dll4/Notch signalling to control angiogenic sprouting. Increasing knowledge of the mechanisms regulating (lymph)angiogenesis should enable the development of better agents to combat metastasis and the resistance of tumours towards anti-angiogenic treatment, and of pro-(lymph)angiogenic treatment methods for ischaemic diseases and lymphoedema.

VEGFs and receptors involved in angiogenesis versus lymphangiogenesis.

Tumor stroma, consisting of the extracellular matrix and multiple cell types such as immune cells, fibroblasts and vascular cells, contributes to the malignancy of solid tumors by a variety of mechanisms. Intravital imaging by different microscopy techniques, especially by confocal and multi-photon microscopy, has proven to be a powerful method for analyzing the cell-cell and cell-matrix interactions in the dynamic tumor microenvironments. Intravital imaging has fostered the acquisition of data on parameters such as motility of different cell types in distinct tumor regions or manipulated with defined challenges, kinetics of tumor cell killing by T cells or macrophage-assisted tumor cell extravasation, functionality of the vasculature, protease activity and metabolic state. Achieving the direct observation of intact tumors offered by intravital imaging provides unique insights into tumor biology that will continue to deepen our understanding of the processes leading to malignancy and of the ways they can be targeted.

Intravital imaging of stromal cell dynamics in tumors.

The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28-30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.

Matrix metalloproteinase-9 and stromal cell-derived factor-1 act synergistically to support migration of blood-borne monocytes into the injured spinal cord.

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.

Effects of angiopoietin-2-blocking antibody on endothelial cell-cell junctions and lung metastasis.

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

Marja Lohela

Department of Anatomy

University of California

Real-time Imaging of Myeloid Cells Dynamics in Apc^Min/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Marja Lohela

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Anatomy

University of California

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Department of Anatomy

Real-time Imaging of Myeloid Cells Dynamics in Apc^Min/+ Intestinal Tumors by Spinning Disk Confocal Microscopy