Increased Blood Vessel Density Research Articles

Deficiency of bone marrow β3‐integrin enhances non‐functional neovascularization

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

The Addition of Bevacizumab (B) to Lenalidomide and Low Dose Dexamethasone Does Not Significantly Increase Response in Relapsed or Refractory Multiple Myeloma (NCI#7317).

Abstract 3885Poster Board III-821The interaction between malignant plasma cells and non malignant bone marrow microenvironment has been recognized as both an important feature of MM propagation and a therapeutic target. One critical pathway appears to involve neoangiogenesis as evidenced by increased blood vessel density in areas of myeloma cell proliferation. The potent anti MM compound lenalidomide may work partly through down regulation of VEGF expression by bone marrow stromal cells. We hypothesized that the addition of the VEGF-A receptor inhibitor, bevacizumab, in combination with low dose weekly dexamethasone, would provide more complete blockade of VEGF activation and boost the response rate in MM patients with relapsed or refractory disease over lenalidomide and dexamethasone alone. ELIGIBILTYRelapsed or refractory MM patients (pts), failing >1 prior therapy, and no previous exposure to lenalidomide, measurable M protein in serum and/or urine, no current history of unstable cardiovascular disease or uncontrolled thrombosis, no therapy for 28 days, and no contraindication to aspirin. METHODSPts received 4 week cycles consisted of lenalidomide 25 mg PO d1–21, bevacizumab 10 mg/kg IV over 2 hours every 2 weeks, and dexamethasone (D) 40 mg PO q week. All pts received aspirin 325 mg PO daily. Prior to therapy pts underwent bone marrow biopsy and aspirate. MM and stromal cells were isolated for analysis of STAT 3 activation to assess treatment effect on stromal/MM cell interactions. Plasma VEGF, VEGFR-1 and MIP-1α levels were measured at baseline and after 2 cycles. Clinical responses were assessed using IBMTR criteria every 2 cycles. Pts continued on treatment until progression or toxicity. This study was designed as a Simon minimax two-stage design with 19 pts, followed by 14 additional pts with 10 or more responders out of the initial 19 to detect an objective response rate of 70% with power 0.9 from 45% at a significance level of 0.05. RESULTSAs of this reporting, 31 pts have been enrolled, ages 41–89, with median number of previous regimens 3 (range 1–7), previous transplant 27%. Three pts are considered unevaluable as they were taken off study before 2 cycles, two due to GI perforation and one pt due to rapidly progressive disease during first week of therapy. One pt is too early for evaluation. Twenty-seven pts have completed >4 cycles and have been evaluated for response. Responses are as follows: Complete response-15% (n=4), Partial response 56% (n=15), stable disease 19% (n=5), and progressive disease 11% (n=3). Overall response rate of 70% (a 95% confidence interval 50%–86%) is not significantly different from that reported by Weber et al (NEJM 357:2133) of 61% (a 95% CI 53%–68%) in patients receiving lenalidomide and high dose D. However, overall grade 3/4 toxicities were significantly less than those reported by Weber. Grade 3/4 toxicities included DVT in 3 patients (all were on aspirin but received erythropoiesis stimulating agents), and 2 pts developed shortness of breath attributed to B, which resolved after discontinuation of the drug; 3 pts developed atrial fibrillation. No patient developed hypertension or proteinuria. Most required a dose reduction of lenalidomide due to fatigue. Analysis of STAT3 DNA-binding in MM cells alone or in co-culture with MM-BMSCs revealed variable levels of constitutive STAT3 activity and enhanced activity in co-culture. No additional effect of bevacizumab + lenalidomide on constitutive STAT3 activity was observed. Patients with no constitutive STAT3 activity were those achieving CR. CR was also associated with low plasma MIP-1α and VEGF levels. CONCLUSIONSThe combination of B added to lenalidomide and low dose D has activity in relapsed and refractory myeloma. The initial 70% response rate is not statistically superior to the 60% response rate reported by Weber et al in previously treated MM pts receiving lenalidomide and high dose D. Toxicities of this regimen are predictable from the expected side effect profile of each drug but manageable. The use of ESAs may contribute to the development of DVT, and the protocol was modified to preclude their use. Low levels of constitutive STAT-3 activation and plasma MIP-1α predict for response in this trial. AcknowledgmentsSupported by the U Wisc Carbone Cancer Center (P30 CA14520), Wisconsin Oncology Network, and NCI Translational Research Initiative 25×5097. Genentech Lab and Celgene Corp provided B and lenalidomide for correlative studies. Disclosures:Callander:Millenium: Research Funding. Off Label Use: bevacizumab for multiple myeloma.

Direct Chemical Cross-Linking of Platelet-Derived Growth Factor-BB to the Demineralized Bone Matrix Improves Cellularization and Vascularization

In previous studies, we have described the use of demineralized bone matrix (DBM) as a carrier for the localized delivery system of growth factors in vitro and in vivo. The aim of the present work was to develop a direct chemical approach to immobilize the platelet-derived growth factor-BB (PDGF-BB) on DBM with cross-linking reagents. The amount of PDGF-BB covalently immobilized on DBM was significantly increased. The increased proliferation of fibroblasts demonstrated that the biological activity of PDGF-BB was not significantly reduced by cross-linking. Compared with control groups, there was a statistically significant increase in blood vessel density in the PDGF-C-DBM group after having been subcutaneously implanted into the dorsal side of the rats. The surface bioactivity of scaffolds on stimulation cell and new blood vessel invasion was improved. Therefore, the direct chemical cross-linking approach could be used to retain growth factors on collagen scaffolds effectively to develop functional biomaterials.

Abstract 5052: Attenuation of Ventricular Remodeling and Improvement of Left Ventricular Systolic Function by Injection of Human Fetal Cardiomyocyte Progenitor Cells After Acute Myocardial Infarction

Introduction: The human cardiomyocyte progenitor cell (hCMPC) is part of a population of stem cells located in the human heart. Isolates of hCMPCs from human fetal hearts showed differentiation into functional cardiomyocytes in vitro. In the present study we investigated the potential of hCMPCs to restore ischemic myocardium and improve left ventricular (LV) function in a myocardial infarction (MI) model in mice. Methods: MI was induced in immune-compromised NOD/scid mice, immediately followed by injection of hCMPCs labeled with eGFP (hCMPC group) or vehicle only (control group). From sham operated mice we obtained reference values. Cardiac performance was measured 2 and 14 days after MI using 9.4 Tesla MRI. LV pressure-volume measurements were performed at day 15 followed by extensive immunohistological analysis. Results: Animals injected with hCMPCs after MI demonstrated a significantly higher LV ejection fraction, a lower LV end-systolic volume and a smaller ô (relaxation time constant), compared to animals from the control group (by 17%, by 22% and by 24% respectively, P<0.05). A trend towards a lower LV end-diastolic volume was also observed, although this did not reach significance (P=0.14). Both the infarct zone and border zone of the hCMCP group showed a significant increase in total blood vessel density (6.67%±0.85% and 9.13%±0.46%) compared to control (3.35%±1.13% and 4.31%±1.08%, P<0.01 in either zone). The infarct and border zone of the hearts injected with hCMPCs also had a diminished collagen type III density (1.07±0.06 and 1.11±0.07), compared to the control group (1.45±0.05 and 1.51±0.08, P<0.01 in either zone). Furthermore, nuclear PCNA expression indicated an increased rate of mitosis in the infarct (37.76%±2.23%) and border zone (48.84%±2.07%) compared to the infarct (25.48%±4.83%, P<0.01) and border zone (32.03%±1.03%, P<0.01) of the control group. Conclusions: Fetal hCMPCs engraft into infarcted myocardium where they improve LV systolic function and attenuate ventricular remodeling. Increased blood vessel density, elevated mitosis rate and decreased collagen III deposition may be involved in this process. These results indicate that hCMPCs are promising candidates for cell-based therapy.

MicroRNA-92a Controls Angiogenesis and Functional Recovery of Ischemic Tissues in Mice

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to target messenger RNAs (mRNAs), leading to translational repression or degradation. Here, we show that the miR-17approximately92 cluster is highly expressed in human endothelial cells and that miR-92a, a component of this cluster, controls the growth of new blood vessels (angiogenesis). Forced overexpression of miR-92a in endothelial cells blocked angiogenesis in vitro and in vivo. In mouse models of limb ischemia and myocardial infarction, systemic administration of an antagomir designed to inhibit miR-92a led to enhanced blood vessel growth and functional recovery of damaged tissue. MiR-92a appears to target mRNAs corresponding to several proangiogenic proteins, including the integrin subunit alpha5. Thus, miR-92a may serve as a valuable therapeutic target in the setting of ischemic disease.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity

AbstractThe development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDHhi) activity, a progenitor cell function conserved between several lineages. BM ALDHhi cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDHhi cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDHhi cells, mice transplanted with purified ALDHhi cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDHhi cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDHhi cell engraftment stimulated endogenous revascularization. Thus, human BM ALDHhi cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.

Sca-1 + Stem Cell Survival and Engraftment in the Infarcted Heart

We report that elevated connexin-43 (Cx-43) in stem cells preconditioned with insulin-like growth factor-1 (IGF-1) is cytoprotective and reprograms the cells for cardiomyogenic differentiation. Sca-1+ cells were preconditioned with 100 nmol/L IGF-1 for 30 minutes followed by 8 hours of oxygen glucose deprivation to assess the cytoprotective effects of preconditioning. LDH release assay, cytochrome c release, and mitochondrial membrane potential assay showed improved survival of preconditioned Sca-1+ cells under oxygen glucose deprivation compared with nonpreconditioned Sca-1+ cells via PI3K/Akt-dependent caspase-3 downregulation. We observed PI3K/Akt-dependent upregulation of cardiac-specific markers including MEF-2c (2.5-fold), GATA4 (3.1-fold), and Cx-43 (3.5-fold). Cx-43 inhibition with specific RNA interference reduced cell survival under oxygen glucose deprivation and after transplantation. In vivo studies were performed in a female rat model of acute myocardial infarction (n=78). Animals were grouped to receive intramyocardially 70 microL Dulbecco modified Eagles medium without cells (group 1) or containing male 1 x 10(6) nonpreconditioned Sca-1+ cells (group 2) or preconditioned Sca-1+ (group 3) cells labeled with PKH26. Survival of the preconditioned Sca-1+ cells was 5.5-fold higher in group 3 compared with group 2 at 7 days after transplantation. Confocal imaging after actinin and Cx-43 specific immunostaining showed extensive engraftment and myogenic differentiation of preconditioned Sca-1+ cells. Compared with group 2, group 3 showed increased blood vessel density (22.3+/-1.7 per microscopic field; P<0.0001) and attenuated infarction size (23.3+/-3.6%; P=0.002). Heart function indices including ejection fraction (56.2+/-3.5; P=0.029) and fractional shortening (24.3+/-2.1; P=0.03) were improved in group 3 compared with group 2. Preconditioning with IGF-1 reprograms Sca-1+ for prosurvival signaling and cardiomyogenic differentiation with an important role for Cx-43 in this process.

Superficial ovarian cortex vascularization is inversely related to the follicle reserve in normal cycling ovaries and is increased in polycystic ovary syndrome

The superficial ovarian cortex constitutes the micro-environment where resting and early growing follicles reside. As small follicles do not possess an independent capillary network, both their survival and early growth depend on their proximity to the cortical vessels. Little is known about the possible changes in superficial ovarian cortex vascularization in normal women throughout reproductive life or in pathological conditions such as polycystic ovary syndrome (PCOS) involving abnormal early follicle growth. We studied the vascularization of the superficial and deep cortical stroma (DCS) in normal cycling ovaries from 21 to 50 years of age and in infertile women with PCOS. We used archival ovarian samples and specific CD34 immunostaining to determine blood vessel density and to analyse correlation with age and with the ovarian follicle reserve. Normal cycling ovaries showed an age-related increase in the superficial cortical stroma vascularization that was inversely correlated with the density of small (primordial and primary) follicles. In contrast, blood vessel density in the DCS significantly decreased in women aged >or=40 years. Ovaries from PCOS showed a 2-fold increase in blood vessel density in both superficial cortical stroma and DCS with respect to age-matched controls. The increased vascularization of the superficial cortical stroma in normal ovaries in relation to age and in ovaries from PCOS could have profound effects on cortical metabolic rate, primordial follicle survival/activation and early follicle growth, and may underline changes in follicle dynamics in mid-aged women and in PCOS.

Activated Protein C Improves Ischemic Flap Survival and Modulates Proangiogenic and Antiinflammatory Gene Expression

Flap necrosis remains a major complication in reconstructive surgery. The authors evaluated whether systemic activated protein C, a natural serum anticoagulant with anti-inflammatory, proangiogenic, and cytoprotective properties, can improve ischemic skin flap survival. Cranially based dorsal cutaneous flaps were elevated on 44 rats. Animals received intravenous injections of activated protein C (25 microg/kg) or saline. Rats were divided into three groups depending on the timing of the first injection: postoperative (45 minutes postoperatively, n = 12), late preoperative (45 minutes preoperatively, n = 5), and early preoperative (3 hours preoperatively, n = 5). In all groups, second and third injections were performed at 3 and 24 hours postoperatively. Flap survival was measured on day 7. Histological and real-time polymerase chain reaction specimens were collected on days 2 and 7 and at 3 and 24 hours, respectively. Postoperative activated protein C improved flap survival (68.9 +/- 4.3 percent) compared with control treatment (39.3 +/- 1.5 percent; p < 0.001). Late preoperative treatment produced diffuse flap hemorrhage. Early preoperative activated protein C injection produced near-complete flap survival (96.1 +/- 1.1 percent for activated protein C versus 50.1 +/- 3.3 percent for control; p < 0.001). Significantly fewer inflammatory cells, improved muscle viability, and increased blood vessel density were observed in activated protein C-treated versus control rats. Activated protein C treatment significantly reduced mRNA levels of intercellular adhesion molecule-1 and tumor necrosis factor-alpha, while increasing levels of Egr-1, vascular endothelial growth factor receptor 2, and Bcl-2. Systemic activated protein C modulates genes involved in angiogenesis, inflammation and apoptosis and improves ischemic flap survival.

Netrin-4 Enhances Angiogenesis and Neurologic Outcome after Cerebral Ischemia

Functional recovery after cerebral ischemia is mediated by the regeneration of vascular networks and the restoration of synaptic architecture. Netrins have been implicated in neuronal pathfinding and angiogenesis. In this study, we investigated the expression of Netrin-4 and its putative receptors, deleted in colorectal cancer (DCC), Unc5A, and Unc5B after distal middle cerebral artery occlusion in mice. Netrin-4 protein was also administered intracerebroventricularly to examine its effect on angiogenesis and behavioral recovery. Netrin-4 protein was highly upregulated in the ischemic core as soon as 1 day after cerebral ischemia, with subsequent downregulation after 1 week. Its expression was limited to the area of blood-brain barrier damage and was seen on both blood vessels and astrocytic foot processes. Although there was not a significant upregulation of the putative Netrin-4 receptor Unc5A and Unc5B, there was a significant increase in expression of the DCC receptor on neuronal processes in the peri-infarct cortex. Intracerebroventricular administration of Netrin-4 into the ischemic brain increased blood vessel density, endothelial proliferation, and improved behavioral recovery at 1 week after stroke, but did not have an effect on blood-brain barrier permeability or infarct size. These findings suggest that Netrin-4 may improve poststroke functional recovery by enhancing blood vessel proliferation.

Human angiogenic cell precursors restore function in the infarcted rat heart: A comparison of cell delivery routes

We recently isolated angiogenic cell precursors (ACPs) from human blood, which can induce angiogenesis in vitro. In the present study, we used a nude rat model of ischaemic cardiomyopathy to compare the efficacy of intramyocardial and intracoronary ACP implantation, and to evaluate effects on cardiac function, scar size and angiogenesis. Adult nude rats underwent coronary artery ligation. Six days later, ACPs (characterized in vitro prior to implantation) or culture media were injected directly into the ischaemic myocardial region or into the coronary artery via the aorta. Cardiac function was measured by echocardiography prior to and at 2 and 4 weeks after implantation. Scar morphology, cell engraftment, and myocardial angiogenesis were evaluated at 4 weeks. Two and four weeks after implantation, cardiac function declined in both of the control groups but improved in both the intramyocardial and intracoronary ACP groups. Significant reductions in myocardial scar area were only observed in the intramyocardial ACP group, while increases in blood vessel density, which were observed in all ACP recipients, were greatest in the intracoronary ACP group. Human ACPs, delivered via intramyocardial or intracoronary injection, engrafted into damaged cardiac tissue and improved cardiac function within 4 weeks through effects on scar morphology and blood vessel formation.

Type I collagen expression contributes to angiogenesis and the development of deeply invasive cutaneous melanoma

Tumors are complex tissues composed of neoplastic cells, soluble and insoluble matrix components and stromal cells. Here we report that in melanoma, turn-over of type I collagen (Col(I)), the predominant matrix protein in dermal stroma affects melanoma progression. Fibroblasts juxtaposed to melanoma cell nests within the papillary dermis display high levels of Col(I) mRNA expression. These nests are enveloped by collagen fibers. In contrast, melanoma-associated fibroblasts within the reticular dermis express Col(I) mRNA at a level that is comparable to its expression in uninvolved dermis and reduced amount of collagen protein can be observed. To determine the significance of Col(I) expression in melanoma, we pharmacologically inhibited its transcription in a porcine cutaneous melanoma model by oral administration of halofuginone. When administered before melanoma development, it reduced melanoma incidence and diminished the transition from microinvasive toward deeply invasive growth by limiting the development of a tumor vasculature. Whereas invasive melanoma growth has been correlated with increased blood vessel density previously, our data for the first time demonstrate that the proangiogenic effect of Col(I) expression by fibroblasts and vascular cells precedes the development of invasive melanomas in a de novo tumor model.

Phase II Trial of Bevacizumab Combined with Low Dose Dexamethasone and Lenalidomide (BEV/REV/DEX) for Relapsed or Refractory Myeloma (MM).

The interaction between malignant plasma cells and non malignant bone marrow microenvironment has been recognized as both an important feature of MM propagation and a therapeutic target. One critical interaction appears to involve angiogenesis as evidenced by increased blood vessel density in areas of myeloma cell proliferation. The potent anti MM compound lenalidomide may work partly through down regulation of VEGF expression by bone marrow stromal cells. We hypothesized that the addition of the VEGF-A receptor inhibitor, bevacizumab, in combination with low dose weekly dexamethasone, would provide more complete blockade of VEGF activation and translate to increased activity in MM patients with relapsed or refractory disease.ELIGIBILTY: relapsed or refractory MM patients (pts), failing >1 therapy, with no previous exposure to lenalidomide, measurable M protein in serum and/or urine, no current history of unstable cardiovascular disease or uncontrolled thrombosis, no therapy for ≥28 days, and no contraindication to aspirin.METHODS: Each 4 week cycle consisted of lenalidomide 25 mg PO d1–21, bevacizumab 10 mg/kg IV over 2 hours every two weeks, and dexamethasone 40 mg PO q week. All pts received aspirin 325 mg PO daily. Prior to therapy pts underwent bone marrow biopsy and aspirate. MM and stromal cells were isolated for analysis of STAT 3 activation to assess the treatment's effect on stromal/MM cell interactions. Clinical responses were assessed using IBMTR criteria.RESULTS: 17 pts have been enrolled, ages 53––89, with median number of previous regimens 3 (range 1–6). Two pts were taken off study during the first cycle, one due to GI perforation occurring d 6 of therapy, and one pt due to rapidly progressive disease during first week of therapy. Ten pts have completed ≥ 4 cycles and can be evaluated for response. Seven of 10 pts achieved a PR after a median of two cycles and have maintained that response. One pt progressed after completing one cycle, one pt progressed after 5 cycles, and one pt had stable disease after 6 cycles. Expected grade 3 toxicities included DVT in 2 patients (both of whom were on aspirin but received erythropoiesis stimulating agents) and 2 pts developed shortness of breath attributed to bevacizumab, with resolved after discontinuation of the drug; one patient developed atrial fibrillation which spontaneously converted. No patient developed hypertension or proteinuria. Most required a dose reduction of lenalidomide due to fatigue. Analysis of STAT3 DNA-binding in MM cells alone or in co-culture with MM-BMSCs revealed variable low levels of constitutive STAT3 activity and enhanced activity in co-culture. No additional effect of bevacizumab + revlimid on constitutive STAT3 activity was observed.CONCLUSIONS: the combination of lenalidomide, bevacizumab and low dose dexamethasone has activity in relapsed and refractory myeloma. The initial 70% response rate compares favorably with the 58% response rate reported by Stadtmauer et al (Blood 108:3552) in previously treated MM pts receiving lenalidomide and high dose dexamethasone. Toxicities of this regimen are predictable but manageable. The use of ESAs may contribute to the development of DVT and the protocol has been modified to preclude their use.

ULTRASONOGRAPHIC DESCRIPTION OF CANINE MASTITIS

Ultrasonographic images were acquired of the mammary glands of 40 bitches with physiologically lactating (n = 20) or inflamed glands (n = 20). Echogenicity, structure, homogeneity, thickness, and distinguishability of each tissue layer were assessed. Additionally, overall echogenicity was noted. In the normal lactating gland, different tissues could be differentiated easily. The parenchyma was, without exception, separated from adjacent tissues and was visible as medium echogenic tissue with a coarse-grained structure. The tissue always had some echogenic lines and anechoic areas and was slightly heterogeneous. The loss of distinct layering of the tissue was characteristic of an inflamed mammary gland and inflamed regions had reduced echogenicity. Additionally in five bitches with mastitis, the ultrasound examination was repeated five times for documentation of the progress of the illness and associated changes, supplemented with a color Doppler sonogram to assess changes in blood vessel density. Information from the examinations carried out via B-mode did not allow treatment success to be predicted. Two bitches with reduced blood vessel density centrally had a poor outcome whereas three bitches with increased blood vessel density had a good outcome. Thus, Doppler sonography might be a useful tool to obtain information of the prognosis in acute canine mastitis.

TIMP-3 deficiency accelerates cardiac remodeling after myocardial infarction

The activity of TIMP-3, a natural tissue inhibitor of matrix metalloproteinases (MMPs), is decreased in the failing heart. This study evaluated the response to coronary ligation of cardiac structure, function, and matrix remodeling in wild-type (WT) mice, and those deficient in TIMP-3 ( timp-3 −/− ). The coronary artery was ligated in timp-3 −/− and age-matched WT mice. At various time points over the following 28-day period, left ventricular structure and function (by echocardiography, pressure–volume measurements and morphometry), MMP levels and activity, blood vessel density, cell proliferation, apoptosis, matrix structure, and inflammatory cytokine levels were assessed in both groups. After ligation, mortality was significantly greater in timp-3 −/− than in WT mice. Morphometry and echocardiography demonstrated no difference in heart size or function prior to ligation; however, the progression of left ventricular systolic dysfunction was accelerated in timp-3 −/− mice at 7, 14 and 28 days after infarction compared to WT controls. Left ventricular dilatation, gelatinase MMP activity, and TNF-α levels were significantly greater in timp-3 −/− than in WT mice at different times after ligation. By histological evaluation, timp-3 −/− mice exhibited significantly increased blood vessel density, cell proliferation, and apoptosis in the infarct area, and reduced collagen content in the viable remote myocardium compared to WT mice at 7 and 14 days after ligation. TIMP-3 deficiency accelerated maladaptive cardiac remodeling after a myocardial infarction by promoting matrix degradation and inflammatory cytokine expression. This study supports further investigations to determine whether such remodeling could be reduced by augmenting TIMP-3 expression in the infarcted myocardium.

Angiopoietin‐1 for myocardial angiogenesis: A comparison between delivery strategies

We compare the effectiveness of direct adenoviral angiopoietin-1 (Ad-Ang-1) injection with transplantation of skeletal myoblasts (SkMs) over-expressing angiopoietin-1 (Ang-1) for angiogenic response and improvement of heart function in an experimental porcine model of myocardial infarction (MI). Ad-Ang-1 was used for intramyocardial injection or transduction of SkMs. Three weeks after coronary artery ligation in 32 female pigs, animals were grouped to receive multiple intramyocardial injections of DMEM without cells (group-1; n=7), or containing 3 x 10(8)Lac-z labelled SkMs transduced with Ad-Null vector carrying no gene (group-2; n=7), or 1 x 10(10) PFU Ad-Ang-1 (group-3; n=9), or 3 x 10(8)Lac-z labelled SkMs transduced with Ad-Ang-1 (group-4; n=9). The animals were immunosuppressed for 6-weeks. After euthanasia, their heart tissue was processed for histological studies. Extensive survival of Lac-z positive SkMs was observed in and around the infarct 6 and 12-weeks after transplantation. Fluorescent immunostaining for vWF-VIII at 6-weeks revealed increased blood vessel density (x100) in group-4 (p<0.05) as compared with other groups. Regional blood flow (ml/g/min) in the peri-infarct area was improved in group-4 (2.7; p<0.05) as compared with group-1 (1.2+/-0.1), group-2 (1.1+/-0.4) and group-3 (1.7+/-0.1) at 6-weeks. Similarly, ejection fraction was significantly higher in group-4 (49.2+/-5.9%, p=0.03) as compared with group-1 (36.8+/-3%) at 6 weeks. SkMs mediated Ang-1 delivery is associated with improved angiogenic response, regional myocardial perfusion and heart function as compared with direct Ad-Ang-1 administration.

Assessment of angiogenic properties of biomaterials using the chicken embryo chorioallantoic membrane assay

The angiogenic potential of a biomaterial is a critical factor for successful graft intake in tissue engineering. We developed a modified, rapid and reproducible chicken embryo chorioallantoic membrane (CAM) assay to evaluate the ability of biomaterials in inducing blood vessel density. Five biomaterials including one-layer porcine small intestinal submucosa (SIS), two-layer SIS, four-layer vacuum pressed (VP) SIS, polyglycolic acid (PGA) and PGA modified with poly(lactic-co-glycolic acid) (PLGA) were analyzed. A circular section (1.2 mm diameter) of each biomaterial was placed near a group of blood vessels in the CAM. Blood vessels around the biomaterials were captured with black and white images at 96 h post implantation; and the images were subjected to densitometry evaluation. One-layer SIS induced a significant increase in blood vessel density as compared to the cellulose nitrate negative control, and had the greatest increase in blood vessel density as compared to four-layer VP SIS, PGA, or PLGA modified PGA. Although two-layer SIS has enhanced physical structure for surgical manipulation, its induction in blood vessel density was significantly lower than the one-layer SIS. Stripping the SIS proteins or incubating one-layer SIS with neutralizing antibodies against basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) resulted in decreased angiogenesis. Consistent with results obtained from bladder augmentation animal models, these results confirmed that angiogenic growth factors were present in SIS and affected the angiogenic potential of biomaterials. These data also demonstrated that the CAM assay can be used to ascertain methodically the angiogenic potential of biomaterials.

Differential Angiogenic Regulation of Experimental Colitis

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract with unknown multifactorial etiology that, among other things, result in alteration and dysfunction of the intestinal microvasculature. Clinical observations of increased colon microvascular density during IBD have been made. However, there have been no reports investigating the physiological or pathological importance of angiogenic stimulation during the development of intestinal inflammation. Here we report that the dextran sodium sulfate and CD4+CD45RBhigh T-cell transfer models of colitis stimulate angiogenesis that results in increased blood vessel density concomitant with increased histopathology, suggesting that the neovasculature contributes to tissue damage during colitis. We also show that leukocyte infiltration is an obligatory requirement for the stimulation of angiogenesis. The angiogenic response during experimental colitis was differentially regulated in that the production of various angiogenic mediators was diverse between the two models with only a small group of molecules being similarly controlled. Importantly, treatment with the anti-angiogenic agent thalidomide or ATN-161 significantly reduced angiogenic activity and associated tissue histopathology during experimental colitis. Our findings identify a direct pathological link between angiogenesis and the development of experimental colitis, representing a novel therapeutic target for IBD.

Myometrial cells induce angiogenesis and salvage damaged myocardium

Characteristically, uterine myometrial cells (MCs) are proliferative, inducing angiogenesis within the female reproductive organ. We evaluated whether MCs implanted into myocardium could also induce angiogenesis and restore heart function after injury. MCs were isolated from the adult rat uterus and cultured for three studies: 1) Intracellular VEGF levels were measured in MCs cultured with progesterone (10(-11), 10(-9), and 10(-7) M) (n = 6 tests per group). 2) Blood vessel density was evaluated 8 days after MCs (3 x 10(6) or 6 x 10(6)), smooth muscle cells (SMCs), or endothelial cells (n = 6 rats per group) were injected with matrigel into the subcutaneous tissue of adult rats. 3) MCs, SMCs (5 x 10(6)/rat), or media were injected into a transmural scar 3 wk after cryoinjury in rat hearts (n = 12 rats per group), and heart function, blood vessel density, and myocardial scar size and thickness were evaluated 5 wk later. In study 1, cultured MCs expressed VEGF, with levels significantly (P < 0.05) upregulated by progesterone at an optimal dose of 10(-11) M. In study 2, MCs injected into the subcutaneous tissue with matrigel induced significantly more blood vessels, especially large-diameter vessels, than did SMCs or endothelial cells (P < 0.01 for all groups). This angiogenic effect was greatest (P < 0.01) at higher doses of MCs and was enhanced by progesterone (10(-11) M). In study 3, MCs implanted into the injured myocardium increased blood vessel density at the implant area, reduced scar size, and improved cardiac function relative to SMCs and media. Overall, MCs induced angiogenesis in vitro and in vivo, prevented cardiac remodeling, and improved heart functional recovery after cardiac injury.

VEGF Scaffolds Enhance Angiogenesis and Bone Regeneration in Irradiated Osseous Defects

Bone regeneration is challenging in sites where the blood supply has been compromised by radiation. We examined the potential of a growth factor (VEGF) delivery system to enhance angiogenesis and bone formation in irradiated calvarial defects. VEGF-releasing polymers significantly increased blood vessel density and vascular perfusion in irradiated defects and increased bone formation relative to control conditions. Radiation therapy causes damage to tissues and inhibits its regenerative capacity. Tissue injury from radiation is in large part caused by a compromised vascular supply and reduced perfusion of tissues. The aim of this study was to determine if delivery of vascular endothelial growth factor (VEGF) from a biodegradable PLGA (copolymer of D,L-lactide and glycolide) scaffold could enhance neovascularization and bone regeneration in irradiated osseous defects. An isolated area of the calvarium of Fisher rats was irradiated (12 Gy) 2 weeks preoperatively, and two 3.5-mm osseous defects were created in this area, followed by the placement of PLGA scaffolds or VEGF scaffolds (PLGA scaffolds with incorporated VEGF) into the defects. Laser Doppler perfusion imaging was performed to measure perfusion of these areas at 1, 2, and 6 weeks. Implants were retrieved at 2, 6, and 12 weeks, and histologic and muCT analyses were performed to determine neovascularization and bone regeneration. Histological analyses revealed statistically significant increases in blood vessel formation (>2-fold) and function (30%) within the VEGF scaffolds compared with PLGA scaffolds. Additionally, evaluation of bone regeneration through bone histomorphometric and muCT analyses revealed significantly greater bone coverage (26.36 +/- 6.91% versus 7.05 +/- 2.09% [SD]) and increased BMD (130.80 +/- 58.05 versus 71.28 +/- 42.94 mg/cm(3)) in VEGF scaffolds compared with PLGA scaffolds. Our findings show that VEGF scaffolds have the ability to enhance neovascularization and bone regeneration in irradiated osseous defects, outlining a novel approach for engineering tissues in hypovascular environments.