Petri Salven

Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth

The mechanisms by which bone marrow (BM)-derived stem cells might contribute to angiogenesis and the origin of neovascular endothelial cells (ECs) are controversial. Neovascular ECs have been proposed to originate from VEGF receptor 2-expressing (VEGFR-2+) stem cells mobilized from the BM by VEGF or tumors, and it is thought that angiogenesis and tumor growth may depend on such endothelial precursors or progenitors. We studied the mobilization of BM cells to circulation by inoculating mice with VEGF polypeptides, adenoviral vectors expressing VEGF, or tumors. We induced angiogenesis by syngeneic melanomas, APCmin adenomas, adenoviral VEGF delivery, or matrigel plugs in four different genetically tagged universal or endothelial cell-specific chimeric mouse models, and subsequently analyzed the contribution of BM-derived cells to endothelium in a wide range of time points. To study the existence of circulating ECs in a nonmyeloablative setting, pairs of genetically marked parabiotic mice with a shared anastomosed circulatory system were created. We did not observe specific mobilization of VEGFR-2+ cells to circulation by VEGF or tumors. During angiogenesis, abundant BM-derived perivascular cells were recruited close to blood vessel wall ECs but did not form part of the endothelium. No circulation-derived vascular ECs were observed in the parabiosis experiments. Our results show that no BM-derived VEGFR-2+ or other EC precursors contribute to vascular endothelium and that cancer growth does not require BM-derived endothelial progenitors. Endothelial differentiation is not a typical in vivo function of normal BM-derived stem cells in adults, and it has to be an extremely rare event if it occurs at all.

Vascular Endothelial Growth Factors VEGF-B and VEGF-C Are Expressed in Human Tumors

The growth of solid tumors is dependent on angiogenesis, the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is a secreted endothelial-cell-specific mitogen. We have recently characterized two novel endothelial growth factors with structural homology to VEGF and named them VEGF-B and VEGF-C. To further define the roles of VEGF-B and VEGF-C, we have studied their expression in a variety of human tumors, both malignant and benign. VEGF-B mRNA was detected in most of the tumor samples studied, and the mRNA and the protein product were localized to tumor cells. Endothelial cells of tumor vessels were also immunoreactive for VEGF-B, probably representing the binding sites of the VEGF-B polypeptide secreted by adjacent tumor cells. VEGF-C mRNA was detected in approximately one-half of the cancers analyzed. Via in situ hybridization, VEGF-C mRNA was also localized to tumor cells. All lymphomas studied contained low levels of VEGF-C mRNA, possibly reflecting the cell-specific pattern of expression of the VEGF-C gene in the corresponding normal cells. The expression of VEGF-C is associated with the development of lymphatic vessels, and VEGF-C could be an important factor regulating the mutual paracrine relationships between tumor cells and lymphatic endothelial cells. Furthermore, VEGF-C and VEGF-B can, similarly to VEGF, be involved in tumor angiogenesis.

Preexisting Lymphatic Endothelium but not Endothelial Progenitor Cells Are Essential for Tumor Lymphangiogenesis and Lymphatic Metastasis

Endothelial progenitor cells have been shown to contribute to angiogenesis in various tumor models. Here, we have studied the relative contributions of bone marrow (BM)-derived endothelial progenitors and pre-existing lymphatic vessels to tumor lymphangiogenesis. We did not find significant incorporation of genetically marked BM-derived cells in lymphatic vessels during tumor- or vascular endothelial growth factor C-induced lymphangiogenesis. The degree of tumor lymphangiogenesis correlated with lymphatic vessel density in the peritumoral area, and despite tumor lymphangiogenesis, lymphatic metastasis failed to occur in gene-targeted vascular endothelial growth factor C+/− mice that have hypoplasia of the lymphatic network. Our data demonstrate that during tumor lymphangiogenesis and cancer cell dissemination via the lymphatics, the newly formed lymphatic vessels sprout from the pre-existing local lymphatic network with little if any incorporation of BM-derived endothelial progenitor cells.

Somatic Progenitor Cell Vulnerability to Mitochondrial DNA Mutagenesis Underlies Progeroid Phenotypes in Polg Mutator Mice

Somatic stem cell (SSC) dysfunction is typical for different progeroid phenotypes in mice with genomic DNA repair defects. MtDNA mutagenesis in mice with defective Polg exonuclease activity also leads to progeroid symptoms, by an unknown mechanism. We found that Polg-Mutator mice had neural (NSC) and hematopoietic progenitor (HPC) dysfunction already from embryogenesis. NSC self-renewal was decreased in vitro, and quiescent NSC amounts were reduced in vivo. HPCs showed abnormal lineage differentiation leading to anemia and lymphopenia. N-acetyl-L-cysteine treatment rescued both NSC and HPC abnormalities, suggesting that subtle ROS/redox changes, induced by mtDNA mutagenesis, modulate SSC function. Our results show that mtDNA mutagenesis affected SSC function early but manifested as respiratory chain deficiency in nondividing tissues in old age. Deletor mice, having mtDNA deletions in postmitotic cells and no progeria, had normal SSCs. We propose that SSC compartment is sensitive to mtDNA mutagenesis, and that mitochondrial dysfunction in SSCs can underlie progeroid manifestations.

Interleukin-1α promotes angiogenesis in vivo via VEGFR-2 pathway by inducing inflammatory cell VEGF synthesis and secretion

During inflammation, functional changes occur in the vasculature, including extensive endothelial cell mitotic activity and remodeling of capillaries. Interleukin‐1α. (IL‐1α) is a prototypical proinflammatory cytokine. Vascular endothelial growth factor (VEGF) is a strong endothelial cell‐specific mitogen that exerts a pivotal role in angiogenesis under physiological and pathological conditions. We show that IL‐1α stimulates VEGF secretion by human peripheral blood mononuclear cells (PBMNCs) in a dose‐dependent manner. This represents induction of de novo VEGF synthesis, as an induction of VEGF mRNA was observed. Also, the release of VEGF was blocked by cycloheximide. Reverse transcription‐polymerase chain reaction (RT‐PCR) detected four VEGF splice variants in unstimulated and in IL‐1α‐stimulated PBMNCs. in vivo in mice, subcutaneously administered IL‐1α caused a strong local angiogenic response, which was accompanied by an infiltrate of VEGF‐expressing inflammatory cells. The angiogenic effect of IL‐1α was blocked when the mice were treated with VEGF receptor 2 (VEGFR‐2) neutralizing antibodies. VEGFR‐1 blocking antibodies had a marginal inhibitory effect on IL‐1α‐induced angiogenesis. These observations indicate that IL‐1α induces angiogenesis by activating the VEGF‐VEGFR‐2 signaling pathway between inflammatory cells and blood vessel endothelial cells. This novel mechanism of IL‐1α‐action may enhance the shift to angiogenic phenotype in various conditions designated by excessive angiogenesis.

Generation of functional blood vessels from a single c-kit+ adult vascular endothelial stem cell.

Adult vascular endothelial stem cells are shown to reside in the blood vessel wall endothelium. When isolated, these cells are capable of clonal expansion and generate functional blood vessels in vivo.

Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis

Bmx/Etk non-receptor tyrosine protein kinase has been implicated in endothelial cell migration and tube formation in vitro. However, the role of Bmx in vivo is not known. Bmx is highly induced in the vasculature of ischemic hind limbs. We used both mice with a genetic deletion of Bmx (Bmx-KO mice) and transgenic mice expressing a constitutively active form of Bmx under the endothelial Tie-2 enhancer/promoter (Bmx-SK-Tg mice) to study the role of Bmx in ischemia-mediated arteriogenesis/angiogenesis. In response to ischemia, Bmx-KO mice had markedly reduced, whereas Bmx-SK-Tg mice had enhanced, clinical recovery, limb perfusion, and ischemic reserve capacity when compared with nontransgenic control mice. The functional outcomes in these mice were correlated with ischemia-initiated arteriogenesis, capillary formation, and vessel maturation as well as Bmx-dependent expression/activation of TNF receptor 2- and VEGFR2-mediated (TNFR2/VEGFR2-mediated) angiogenic signaling in both hind limb and bone marrow. More importantly, results of bone marrow transplantation studies showed that Bmx in bone marrow-derived cells plays a critical role in the early phase of ischemic tissue remodeling. Our study provides the first demonstration to our knowledge that Bmx in endothelium and bone marrow plays a critical role in arteriogenesis/angiogenesis in vivo and suggests that Bmx may be a novel target for the treatment of vascular diseases such as coronary artery disease and peripheral arterial disease.

The EGFR inhibitor gefitinib suppresses recruitment of pericytes and bone marrow-derived perivascular cells into tumor vessels.

Drugs that target EGFR have established anti-tumor effect and are used in the clinic. Here we addressed whether inhibition of EGFR tyrosine kinase activity by gefitinib in tumor microenvironment affected tumor angiogenesis or vasculogenesis. A syngeneic tumor model of mice with grafted GFP-labeled bone marrow cells was used to analyze the effects of gefitinib on different cellular components of tumor vasculature. To characterize tumor cell-independent stromal effects of EGFR targeting, the mice were injected with B16 melanoma cells not expressing significant quantities of EGFR, and treated with gefitinib for seven days, a period not sufficient for significant reduction in total tumor volume. Numbers of vessels as well as cell surface areas covered by markers of endothelial, pericyte and bone marrow-derived progenitor cells were quantified by image analysis of tumor sections. Quantitative analysis of immunohistochemical data demonstrated that gefitinib decreased the coverage of small CD31-positive vessels with NG2-positive pericytes, as well as reduced the recruitment of perivascular GFP-positive bone marrow-derived progenitor cells within the tumor tissue. These results suggest that inhibition of EGFR activity in tumors has vascular effects in the absence of direct effect on tumor cells. EGFR targeting may lead to suppressed mobilization of pericytes needed for vessel stabilization, as well as of bone marrow-derived perivascular progenitor cells. These findings introduce novel cellular mechanisms by which EGFR targeted drugs may suppress tumor growth.

Endotoxins induce and interferon α suppresses vascular endothelial growth factor (VEGF) production in human peripheral blood mononuclear cells1

Endotoxins, classical activators of innate immune cells, induce systemic inflammation designated by increased microvascular permeability, and, not so infrequently, circulatory shock, organ dysfunction, and death. Vascular endothelial growth factor (VEGF), besides being an endothelial‐cell mitogen, is a strong vascular permeability factor. This study demonstrates that peripheral blood mononuclear cells (PBMNCs) from healthy volunteers cultured in serum‐free medium in the absence of any stimulus release VEGF continuously into culture media. Physiological endotoxin concentrations stimulate this VEGF secretion in a dose‐dependent manner, representing an induction of de novo VEGF production, as an induction of the expression of the major 3.7‐kb VEGF mRNA transcripts was observed, and the release of VEGF was blocked by cycloheximide. Reverse transcription‐polymerase chain reaction also verified the expression of the four VEGF transcripts. Interferon‐α, a modulator of the immune system and an inhibitor of angiogenesis, inhibited VEGF release dose dependently. We conclude that endotoxin promotes VEGF production in PBMNCs. Circulating and emigrating VEGF‐producing PBMNCs may enhance the shift to angiogenic phenotype in a variety of infectious and inflammatory disorders as well as in cancer. VEGF‐producing PBMNCs may be a novel mechanism of tissue edema in systemic inflammation. Inhibition of VEGF production in PBMNCs may be one of the mechanisms of interferon‐α action.

Concise Review: Endothelial Stem and Progenitor Cells and Their Habitats

Recent studies on the stem cell origins of regenerating tissues have provided solid evidence in support of the role of the resident cells, rather than bone marrow‐derived or transplanted stem cells, in restoring tissue architecture after an injury. This is also true for endothelial stem and progenitor cells: local pools exist in the vascular wall, and those cells are the primary drivers of vascular regeneration. This paradigm shift offers an opportunity to rethink and refine our understanding of the multiple therapeutic effects of transplanted endothelial progenitor cells, focusing on their secretome, sheddome, intercellular communicational routes, and other potential ways to rejuvenate and replenish the pool of resident cells. The dynamics of vascular wall resident cells, at least in the adipose tissue, may shed light on the origins of other cells present in the vascular wall—pericytes and mesenchymal stem cells. The fate of these cells in aging and disease awaits elucidation.