Decoding cancer cell death-driven immune cell recruitment: An in vivo method for site-of-vaccination analyses.

Anticancer vaccines have recently received renewed attention for immunotherapy of at least a subset of cancer-types. Such vaccines mostly involve either killed cancer or tumor cells alone, or combinations thereof with specific (co-incubated) innate immune cells. In recent years, the immunogenic characteristics of the dead or dying cancer cells have emerged as decisive factors behind the success of anticancer vaccines. This has amplified the importance of accounting for immunology of cell death while preparing anticancer vaccines. This, in turn, has increased the emphasis on the immune reactions at the site-of-vaccination since the therapeutic efficacy of the killed cancer/tumor cell vaccines is contingent upon the nature and characteristics of these reactions at the site-of-injection. In this article, we present a systematic methodology that exploits the murine ear pinna model to study differential immune cell recruitment by dead/dying cancer cells injected in vivo, thereby modeling the site-of-injection relevant for anticancer vaccines.

Lymphatic Vascular Structures: A New Aspect in Proliferative Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common diabetic microvascular complication and major cause of blindness in working-age adults. According to the level of microvascular degeneration and ischemic damage, DR is classified into non-proliferative DR (NPDR), and end-stage, proliferative DR (PDR). Despite advances in the disease etiology and pathogenesis, molecular understanding of end-stage PDR, characterized by ischemia- and inflammation-associated neovascularization and fibrosis, remains incomplete due to the limited availability of ideal clinical samples and experimental research models. Since a great portion of patients do not benefit from current treatments, improved therapies are essential. DR is known to be a complex and multifactorial disease featuring the interplay of microvascular, neurodegenerative, metabolic, genetic/epigenetic, immunological, and inflammation-related factors. Particularly, deeper knowledge on the mechanisms and pathophysiology of most advanced PDR is critical. Lymphatic-like vessel formation coupled with abnormal endothelial differentiation and progenitor cell involvement in the neovascularization associated with PDR are novel recent findings which hold potential for improved DR treatment. Understanding the underlying mechanisms of PDR pathogenesis is therefore crucial. To this goal, multidisciplinary approaches and new ex vivo models have been developed for a more comprehensive molecular, cellular and tissue-level understanding of the disease. This is the first step to gain the needed information on how PDR can be better evaluated, stratified, and treated.

Pathogen response-like recruitment and activation of neutrophils by sterile immunogenic dying cells drives neutrophil-mediated residual cell killing.

Innate immune sensing of dying cells is modulated by several signals. Inflammatory chemokines-guided early recruitment, and pathogen-associated molecular patterns-triggered activation, of major anti-pathogenic innate immune cells like neutrophils distinguishes pathogen-infected stressed/dying cells from sterile dying cells. However, whether certain sterile dying cells stimulate innate immunity by partially mimicking pathogen response-like recruitment/activation of neutrophils remains poorly understood. We reveal that sterile immunogenic dying cancer cells trigger (a cell autonomous) pathogen response-like chemokine (PARC) signature, hallmarked by co-release of CXCL1, CCL2 and CXCL10 (similar to cells infected with bacteria or viruses). This PARC signature recruits preferentially neutrophils as first innate immune responders in vivo (in a cross-species, evolutionarily conserved manner; in mice and zebrafish). Furthermore, key danger signals emanating from these dying cells, that is, surface calreticulin, ATP and nucleic acids stimulate phagocytosis, purinergic receptors and toll-like receptors (TLR) i.e. TLR7/8/9-MyD88 signaling on neutrophil level, respectively. Engagement of purinergic receptors and TLR7/8/9-MyD88 signaling evokes neutrophil activation, which culminates into H2O2 and NO-driven respiratory burst-mediated killing of viable residual cancer cells. Thus sterile immunogenic dying cells perform 'altered-self mimicry' in certain contexts to exploit neutrophils for phagocytic targeting of dead/dying cancer cells and cytotoxic targeting of residual cancer cells.

A Case of Abnormal Lymphatic-Like Differentiation and Endothelial Progenitor Cell Activation in Neovascularization Associated with Hemi-Retinal Vein Occlusion.

PURPOSE: Pathological vascular differentiation in retinal vein occlusion (RVO)-related neovessel formation remains poorly characterized. The role of intraocular lymphatic-like differentiation or endothelial progenitor cell activity has not been studied in this disease. METHODS: Vitrectomy was performed in an eye with hemi-RVO; the neovessel membrane located at the optic nerve head was removed and subjected to immunohistochemistry. Characterization of the neovascular tissue was performed using hematoxylin and eosin, α-smooth muscle actin, and the pan-endothelial cell (EC) adhesion molecule CD31. The expression of lymphatic EC markers was studied by lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), podoplanin (PDPN), and prospero-related homeobox protein 1 (Prox-1). Potential vascular stem/progenitor cells were identified by active cellular proliferation (Ki67) and expression of the stem cell marker CD117. RESULTS: The specimen contained blood vessels lined by ECs and surrounded by pericytes. Immunoreactivity for LYVE-1 and Prox-1 was detected, with Prox-1 being more widely expressed in the active Ki67-positive lumen-lining cells. PDPN expression was instead found in the cells residing in the extravascular tissue. Expression of the stem cell markers CD117 and Ki67 suggested vascular endothelial progenitor cell activity. CONCLUSIONS: Intraocular lymphatic-like differentiation coupled with progenitor cell activation may be involved in the pathology of neovessel formation in ischemia-induced human hemi-RVO.

Indications of lymphatic endothelial differentiation and endothelial progenitor cell activation in the pathology of proliferative diabetic retinopathy.

PURPOSE: Proliferative diabetic retinopathy (PDR) is characterized by ischaemia- and inflammation-induced neovascularization, but the pathological vascular differentiation in PDR remains poorly characterized. Here, endothelial progenitor and growth properties, as well as potential lymphatic differentiation, were investigated in the neovascular membrane specimens from vitrectomized patients with PDR. METHODS: The expression of pan-endothelial CD31 (PECAM-1), ETS-related gene (ERG), α-smooth muscle actin (α-SMA), and stem/progenitor cell marker CD117 (c-kit) and cell proliferation marker Ki67 was investigated along with the markers of lymphatic endothelial differentiation (vascular endothelial growth factor receptor (VEGFR)-3; prospero-related homeobox gene-1 (Prox-1), lymphatic vessel endothelial receptor [LYVE)-1 and podoplanin (PDPN)] by immunohistochemistry. Lymphocyte antigen CD45 and pan-macrophage marker CD68 were likewise investigated. RESULTS: All specimens displayed CD31, ERG and α-SMA immunoreactivity in irregular blood vessels. Unexpectedly, VEGFR3 and Prox-1 lymphatic marker positive vessels were also detected in several tissues. Prox-1 was co-expressed with CD117 in lumen-lining endothelial cells and adjacent cells, representing putative endothelial stem/progenitor cells and pro-angiogenic perivascular cells. Immunoreactivity of CD45 and CD68 was detectable in all investigated diabetic neovessel specimens. PDPN immunoreactivity was also detected in irregular lumen-forming structures, but these cells lacked CD31 and ERG that mark blood and lymphatic endothelium. CONCLUSIONS: Although the inner part of human eye is physiologically devoid of lymphatic vessels, lymphatic differentiation associated with endothelial stem/progenitor cell activation may be involved in the pathogenesis of human PDR. Further studies are warranted to elucidate whether targeting lymphatic factors could be beneficial in the treatment of patients with the sight-threatening forms of DR.

[Stem cells in cancer]. / Kantasolut syövässä.

The adult body contains undifferentiated stem cells of various tissues which, when necessary, will divide and produce new differentiated cells to replace dying cells, for example. Malignant tumors contain also less common populations of cancerous cells having a higher capacity than other cancerous cells to produce progeny. Cell structure molecules and regulatory mechanisms similar to those observed on normal stem cells have been detected on these cancer stem cells. Production and self-regeneration of stem cell progeny are subjects, the regulation of which is hoped to reveal mechanisms providing a target for intervention to enable the control of the growth of cancer cells and healthy auxiliary tissue of the tumor.

TIM-family molecules in embryonic hematopoiesis: fetal liver TIM-4(lo) cells have myeloid potential.

Trans-membrane (or T cell) immunoglobulin and mucin (TIM) molecules are known regulators of immune response whose function in hematopoiesis is unknown. Earlier, we found that tim-1 and tim-4 are expressed by CD45(+) cells in the para-aortic region of chicken embryo. Because the para-aortic region is a known site for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) differentiation and expansion, we hypothesize that TIM molecules have a role in hematopoiesis. To study this role further, we analyzed TIM expression more precisely in chicken para-aortic region and mouse fetal liver hematopoietic cells. Additionally, we examined the hematopoietic potential of TIM-4(+) mouse fetal liver cells with a colony-forming assay. tim-1 gene expression was detected in chicken and mouse embryos in the aorta-gonads-mesonephros-region at the time of HSC emergence, whereas tim-3 mRNA was widely expressed in different tissues. tim-4 expression was restricted to fetal liver CD45(+)F4/80(+) cells. Moreover, two TIM-4(+) populations were distinguished: F4/80(hi)TIM-4(hi) and F4/80(lo)TIM-4(lo). F4/80(hi)TIM-4(hi) cells had no hematopoietic potential and were morphologically similar to mature macrophages, suggesting that they are yolk sac-derived macrophages. Instead, many of the F4/80(lo)TIM-4(lo) cells were c-kit(+) and Sca-1(+) and had primitive morphology and multilineage colony-forming ability. In addition, F4/80(lo)TIM-4(lo) cells included a considerable population expressing ER-MP12, a known marker for macrophage colony-forming cells and other myeloid progenitors. We conclude that TIM molecules are expressed in embryonic hematopoietic tissues in chicken and mouse and that in fetal liver, TIM-4 is expressed by myeloid progenitor cells.

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.

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

In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin-CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth.

Dual action of TGF-ß induces vascular growth in vivo through recruitment of angiogenic VEGF-producing hematopoietic effector cells.

The role of Transforming growth factor ß (TGF-ß) as a regulator of blood vessel endothelium is complicated and controversial, and the mechanisms by which TGF-ß is able to induce angiogenesis in vivo are not well understood. Here we show that TGF-ß causes in vivo a massive recruitment of tissue infiltrating hematopoietic cells. Concurrently, TGF-ß induces strong vascular endothelial growth factor (VEGF) production in the recruited hematopoietic cells, resulting in activated angiogenesis and vascular remodeling. TGF-ß also promoted abnormalities of α-smooth muscle actin-expressing pericytes on angiogenic capillaries. TGF-ß-induced angiogenic effect was inhibited by a systemic treatment with VEGF-neutralizing antibodies. When studied in isolated human hematopoietic cells, physiological concentrations of TGF-ß stimulated VEGF mRNA and protein expression in a dose- and time-dependent manner. This induction was p38 and p44/p42 mitogen activated kinase dependent. p38 and p44/p42 activation was also observed in vivo in TGF-ß-treated angiogenic murine tissues. Taken together, our results provide a dual action mechanism by which TGF-ß promotes angiogenesis in vivo via recruitment of paracrine VEGF-expressing hematopoietic effector cells. This mechanism may activate vascular growth and remodeling during inflammatory conditions and tumor growth when TGF-ß activity is upregulated.

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.

High serum angiogenin at diagnosis predicts for failure on long-term treatment response and for poor overall survival in non-Hodgkin lymphoma.

BACKGROUND: Angiogenin is a potent inducer of angiogenesis. We prospectively evaluated the prognostic significance of serum angiogenin from 204 consecutive non-Hodgkin lymphoma (NHL) patients diagnosed and treated in a single institution. METHODS: Serum angiogenin, VEGF, and bFGF concentrations at diagnosis were determined using a quantitative sandwich enzyme immunoassay technique. Kaplan-Meier survival curves were compared by the log-rank test. Multivariate survival analyses were performed using the parametric model of Weibull and the non-parametric proportional hazards model of Cox. RESULTS: Patients with a high serum angiogenin at diagnosis (>median; 401 ng/ml) had significantly lower 5-year survival rate than those with a low (≤ median) angiogenin (42% versus 63%, respectively; P = 0.0073). Serum angiogenin provided additional information to the International Prognostic Index (IPI) identifying a subgroup (serum angiogenin >median and IPI>1) with very poor prognosis (5-year survival 19%, P < 0.0001). In receiver operating characteristic (ROC) analyses the accuracy of the IPI to correctly classify patients with favourable or poor survival was improved from fair to good by complementing the IPI with serum angiogenin concentration. With patients who initially achieved complete response (CR) after chemotherapy, a high angiogenin at diagnosis (>median; relative risk (RR) 2.38; P = 0.0077) and an advanced tumour stage (III-IV; RR 2.41; P = 0.0087) were the only independent predictors for patients with unfavourable outcome although first responding well to therapy. CONCLUSIONS: We conclude that elevated serum angiogenin surfaced as an independent predictor for failure in long-term treatment response and for poor overall survival in a series of 204 NHL patients, and might thus also complement the IPI in identifying the patients with particularly aggressive and/or treatment resistant disease.

Stem cells in tumor angiogenesis.

Contribution from diverse tissue-specific stem cell types is required to create the cell populations necessary for the activation of angiogenesis and neovascular growth in cancer. Bone marrow (BM)-derived circulating endothelial progenitors (EPCs) that would differentiate to bona fide endothelial cells (ECs) were previously believed to be necessary for tumor angiogenesis. However, numerous recent studies demonstrate that EPCs are not needed for tumor angiogenesis and indicate EPCs to be artifactual rather than physiological. It is evident that tumor infiltrating hematopoietic cells produced by BM-residing hematopoietic stem cells (HSCs) may contribute to tumor angiogenesis in a paracrine manner by stimulating ECs or by remodeling the extracellular matrix. Therefore, identification of the various hematopoietic cell subpopulations that are critical for tumor angiogenesis and better understanding of their proangiogenic functions and mechanisms of action have potential therapeutic significance. Stem and progenitor cell subsets for also other vascular or perivascular cell types such as pericytes or mesenchymal/stromal cells may provide critical contributions to the growing neovasculature. Furthermore, we hypothesize that the existence of a yet undiscovered-and largely unsearched-tissue-specific adult vascular endothelial stem cell (VESC) would provide completely novel targeted approaches to block pathological angiogenesis and cancer growth. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

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.

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.

VEGFR-1 and -2 regulate inflammation, myocardial angiogenesis, and arteriosclerosis in chronically rejecting cardiac allografts.

OBJECTIVE: Interplay between inflammation and angiogenesis is important in pathological reparative processes such as arteriosclerosis. We investigated how the two vascular endothelial growth factor receptors VEGFR-1 and -2 regulate these events in chronically rejecting cardiac allografts. METHODS AND RESULTS: Chronic rejection in mouse cardiac allografts induced primitive myocardial, adventitial, and intimal angiogenesis with endothelial expression of CD31, stem cell marker c-kit, and VEGFR-2. Experiments using marker gene mice or rats as cardiac allograft recipients revealed that replacement of cardiac allograft endothelial cells with recipient bone marrow- or non-bone marrow-derived cells was rare and restricted only to sites with severe injury. Targeting VEGFR-1 with neutralizing antibodies in mice reduced allograft CD11b+ myelomonocyte infiltration and allograft arteriosclerosis. VEGFR-2 inhibition prevented myocardial c-kit+ and CD31+ angiogenesis in the allograft, and decreased allograft inflammation and arteriosclerosis. CONCLUSIONS: These results suggest interplay of inflammation, primitive donor-derived myocardial angiogenesis, and arteriosclerosis in transplanted hearts, and that targeting VEGFR-1 and -2 differentially regulate these pathological reparative processes.

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.

Contribution of bone marrow-derived pericyte precursor cells to corneal vasculogenesis.

PURPOSE: Bone-marrow (BM)-derived hematopoietic precursor cells are thought to participate in the growth of blood vessels during postnatal vasculogenesis. In this investigation, multichannel laser scanning confocal microscopy and quantitative image analysis were used to study the fate of BM-derived hematopoietic precursor cells in corneal neovascularization. METHODS: A BM-reconstituted mouse model was used in which the BM from enhanced green fluorescent protein (GFP)-positive mice was transplanted into C57BL/6 mice. Basic fibroblast growth factor (bFGF) was used to induce corneal neovascularization in mice. The vasculogenic potential of adult BM-derived cells and their progeny were tested in this in vivo model. Seventy-two histologic sections selected by systematic random sampling from four mice were immunostained and imaged with a confocal microscope and analyzed with image-analysis software. RESULTS: BM-derived endothelial cells did not contribute to bFGF-induced neovascularization in the cornea. BM-derived periendothelial vascular mural cells (pericytes) were detected at sites of neovascularization, whereas endothelial cells of blood vessels originated from preexisting blood vessels in limbal capillaries. Fifty three percent of all neovascular pericytes originated from BM, and 47% of them originated from preexisting corneoscleral limbus capillaries. Ninety-six percent and 92% of BM-derived pericytes also expressed CD45 and CD11b, respectively, suggesting their hematopoietic origin from the BM. CONCLUSIONS: Pericytes of new corneal vessels have a dual source: BM and preexisting limbal capillaries. These findings establish BM as a significant effector organ in corneal disorders associated with neovascularization.

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

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.