IntroductionIn multiple myeloma (MM), the growth of neoplastic plasma cells is directly regulated by microenvironmental factors in the bone marrow. 1 Of these, neoangiogenesis is thought to have a governing role in MM pathogenesis and progression, indicated by increased bone marrow microvascular density (MVD) in patients that is positively correlated with disease activity and by increased expression of angiogenic factors. [2][3][4][5][6] In fact, increased MVD in myeloma is thought to be largely driven by vascular endothelial growth factor (VEGF), which is secreted by MM and stromal cells in the marrow; these cells also express the receptors for VEGF, including VEGF receptor-2 (kinase insert domain-containing receptor/fetal liver kinase-1 [KDR]). [7][8][9] The production of VEGF by both cell types is increased by interleukin-6 (IL-6), which is produced by stromal cells and provides the survival signal for MM cells; VEGF in turn stimulates production of IL-6 and tumor growth. 10 Targeting of angiogenesis by thalidomide [11][12][13][14] and its more potent immunomodulatory derivatives (IMiDs; Celgene, Warren, NJ) 15,16 is an effective therapeutic strategy against MM in newly diagnosed, relapsed, and refractory patients. Recent data showing a proangiogenic gene expression profile within bone marrow endothelial cells (ECs) of patients with MM suggest that these ECs had undergone an angiogenic switch 17 similar to that observed in solid tumors. 18,19 However, the role of neovascularization in mechanisms involved in MM growth and dissemination requires further elucidation. From the clinical perspective, since MVD may not reflect real-time vascular activity in the marrow, and since MVD determination requires a bone marrow biopsy, a less invasive method that can assess dynamic changes in angiogenesis is needed for gaining insight into the natural course of MM and improving patient management.Growth and dissemination of tumors is supported by neovascularization (new vessel formation), which involves vasculogenesis (de novo tube formation analogous to prenatal vascular development) and angiogenesis (sprouting of new capillary vessels from pre-existing vasculature). Exciting new data show that tumor neovascularization involves recruitment of endothelial progenitor cells (EPCs) from bone marrow and, conversely, that inhibition of EPC recruitment inhibits tumor growth, thus establishing the significance of EPCs in tumor progression. [20][21][22][23][24] Consequently, inhibition of EPCs is a promising treatment modality with benefit both for solid and for liquid tumors. New research also has shown that within human bone marrow and cord blood, EPCs are derived from pluripotential stem cells and from more differentiated hemangioblasts, which are the precursors for both hematopoietic cells and 24 Increased mobilization of CECs and EPCs has been associated with cancer, vascular injury and, in patients with lymphoma, poor prognosis. [36][37][38] In MM, EPCs in peripheral blood have not been characterized, and their role in diseas...

IntroductionThe transforming growth factor-␤ (TGF-␤) family of growth factors mediates vascular development and regulates endothelial responses to mechanical, inflammatory, and hypoxic stress. [1][2][3][4][5][6][7][8][9][10] The important role of TGF-␤ in vascular physiology is indicated by defective vasculogenesis and striking vascular inflammation leading to death in mice null for TGF-␤s, their receptors, or their downstream substrates, the Smad proteins. 3,7,11,12 We recently have shown that exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia (1% O 2 ) selectively up-regulates transcription and expression of TGF-␤2 by as much as 20-fold and induces Smad2, Smad3, and Smad4 to associate with DNA. 9 In vascular endothelium, TGF-␤2, similar to TGF-␤1 and TGF-␤3, is produced in a latent form in which the bioactive, 25-kDa TGF-␤ dimer (mature TGF-␤) is noncovalently bound to its propeptide (also known as latency-associated peptide [LAP]) and is unavailable for binding to TGF-␤ membrane receptors. 1 An important physiologic regulator of TGF-␤ bioactivation is thrombospondin-1 (TSP-1), an extracellular matrix protein that is a member of the TSP family of glycoproteins. [13][14][15] TSP-1, a trimer of disulfide-linked 180-kDa subunits, is secreted from platelet ␣-granules, endothelial cells, and vascular smooth muscle cells, and is deposited in extracellular matrix. 16 Binding of TSP-1 to LAP occurs via amino acid sequence K 412 RFK 415 of TSP-1 and amino acid sequence L 54 SKL 57 of LAP, 15,17 and potentially induces a conformational change in LAP that allows interaction of the 25-kDa mature TGF-␤ peptide with its specific membrane receptors. TSP-1 can activate LAPs associated with both latent TGF-␤1 and -␤2, 15 and similarities reported between TGF-␤1-null and TSP-1-null animals 17,18 suggest that TSP-1-mediated TGF-␤ bioactivation is physiologically significant.Mature TGF-␤ can bind to its type I, type II, and type III cell membrane receptors, the first 2 of which are serine/threonine kinases. 19 Once activated by TGF-␤, the type II receptor transphosphorylates the type I receptor, which then phosphorylates Smad2 or Smad3 (receptor-activated Smads [R-Smads]), which in turn heteromerize with Smad4 (Co-Smad) to translocate to the nucleus. Smad complexes accumulate in the nucleus, where they regulate gene transcription by recruiting transcriptional coactivators or inhibitors to DNA. 19 This cross-talk created by the interplay between Smads and other signaling pathways is largely responsible for the diverse and context-specific effects of the TGF-␤ family of proteins.The Smad signaling pathway was recently shown to interact with the transacting protein complex hypoxia-inducible factor-1 (HIF-1), which is a well-characterized transcription factor complex that regulates hypoxia-driven gene expression. 20,21 HIF-1 binds DNA as a heterodimer of 2 basic helix-loop-helix proteins, HIF-1␣ and the aryl hydrocarbon receptor nuclear translocator (ARNT, or HIF-1␤). 22,23 Under normoxic conditions, HIF-1␣ is ra...