Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A164, we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A–expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A164 also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new “giant” lymphatics generated by VEGF-A164 were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.

SUMMARY:Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is an angiogenic cytokine with potential for the treatment of tissue ischemia. To investigate the properties of the new blood vessels induced by VPF/VEGF, we injected an adenoviral vector engineered to express murine VPF/VEGF 164 into several normal tissues of adult nude mice or rats. A dose-dependent angiogenic response was induced in all tissues studied but was more intense and persisted longer (months) in skin and fat than in heart or skeletal muscle (Յ3 weeks). The initial response (within 18 hours) was identical in all tissues studied and was characterized by microvascular hyperpermeability, edema, deposition of an extravascular fibrin gel, and the formation of enlarged, thin-walled pericyte-poor vessels ("mother" vessels). Mother vessels developed from preexisting microvessels after pericyte detachment and basement membrane degradation. Mother vessels were transient structures that evolved variably in different tissues into smaller daughter vessels, disorganized vessel tangles (glomeruloid bodies), and medium-sized muscular arteries and veins. Vascular structures closely resembling mother vessels and each mother vessel derivative have been observed in benign and malignant tumors, in other examples of pathological and physiological angiogenesis, and in vascular malformations. Together these data suggest that VPF/VEGF has a role in the pathogenesis of these entities. They also indicate that the angiogenic response induced by VPF/VEGF is heterogeneous and tissue specific. Finally, the muscular vessels that developed from mother vessels in skin and perimuscle fat have the structure of collaterals and could be useful clinically in the relief of tissue ischemia. (Lab Invest 2000, 80:99-115).

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a recently described family of ectonucleotidases that differentially hydrolyze the ␥ and ␤ phosphate residues of extracellular nucleotides. Expression of this enzymatic activity has the potential to influence nucleotide P2 receptor signaling within the vasculature. We and others have documented that NTPDase1 (CD39, 78 kd) hydrolyzes both triphosphonucleosides and diphosphonucleosides and thereby terminates platelet aggregation responses to adenosine diphosphate (ADP). In contrast, we now show that NTPDase2 (CD39L1, 75 kd), a preferential nucleoside triphosphatase, activates platelet aggregation by converting adenosine triphosphate (ATP) to ADP, the specific agonist of P2Y 1 and P2Y 12 receptors. We developed specific antibodies to murine NTPDase1 and NTPDase2 and observed that both enzymes are present in the cardiac vasculature; NTPDase1 is expressed by endothelium, endocardium, and to a lesser extent by vascular smooth muscle, while NTPDase2 is associated with the adventitia of muscularized vessels, microvascular pericytes, and other cell populations in the subendocardial space. Moreover, NTPDase2 represents a novel marker for microvascular pericytes. IntroductionNucleoside triphosphate diphosphohydrolases (NTPDases) are a family of ectonucleotidases, previously classified as E-type ATPases, ATPDases, ecto-ATPases, or ecto-apyrases. 1-3 These enzymes differentially hydrolyze the terminal ␥ and ␤ phosphate residues of nucleotides, resulting in the rapid formation of the respective diphosphonucleosides and/or monophosphonucleosides. To date, 6 members of this NTPDase family have been identified. 3-13 NTPDase1, NTPDase2, and NTPDase3 are transmembrane proteins associated with the plasma membrane with an active site facing the extracellular space. 4,5,7,9,14 These NTPDase members have been demonstrated to differ in their substrate specificity. For example, NTPDase1 (CD39 [human] or cd39 [murine]) hydrolyzes both nucleoside triphosphates and diphosphates-eg, adenosine triphosphate (ATP) and adenosine diphosphate (ADP), 4,5 -whereas NTPDase2 (CD39L1) is a preferential nucleoside triphosphatase or ATPase. 7,[14][15][16] Extracellular nucleotides in various forms, and at different concentrations, activate multiple P2 receptors: ionotropic P2X and metabotropic P2Y receptors. [17][18][19][20] While NTPDases would be anticipated to generally terminate P2 receptor agonist signaling, we have also observed that NTPDase1 may prevent receptor desensitization by the catalysis of extracellular nucleotides. 21 NTPDase1 may then also facilitate subsequent responses to "pulses" of nucleotides. In addition, NTPDases in tandem with ecto-5Ј-nucleotidase may facilitate the salvage of nucleotides by the ultimate generation of dephosphorylated forms that are taken up by cells via specific transporters. 1,22 NTPDase1 is the major ectonucleotidase at the luminal surface of blood vessels. 5,21,23,24 By converting the ADP released from activated platelets, NTPDase1 modulates platel...