Biomedical subjects

Alexander L Klibanov

Publications and source records attributed to Alexander L Klibanov.

At least 19 recordsLinked to original sources

PubMed2007

Microparticle adhesive dynamics and rolling mediated by selectin-specific antibodies under flow.

In vitro studies were performed to characterize the relative performance of candidate receptors to target microparticles to inflammatory markers on vascular endothelium. To model the interactions of drug-bearing microparticles or imaging contrast agents with the vasculature, 6 micron polystyrene particles bearing antibodies, peptides, or carbohydrates were perfused over immobilized E- or P-selectin in a flow chamber. Microparticles conjugated with HuEP5C7.g2 (HuEP), a monoclonal antibody (mAb) specific to E- and P-selectin, supported leukocyte-like rolling and transient adhesion at venular shear rates. In contrast, microparticles conjugated with a higher affinity mAb specific for P-selectin (G1) were unable to form bonds at venular flow rates. When both HuEP and G1 were conjugated to the microparticle, HuEP supported binding to P-selectin in flow which allowed G1 to form bonds leading to stable adhesion. While the microparticle attachment and rolling performance was not as stable as that mediated by the natural ligands P-selectin Glycoprotein Ligand-1 or sialyl Lewis(x), HuEP performed significantly better than any previously characterized mAb in terms of mediating microparticle binding under flow conditions. HuEP may be a viable alternative to natural ligands to selectins for targeting particles to inflamed endothelium.

Animals↗

PubMed2006

Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours.

We have shown recently that downregulation of the androgen receptor (AR), one of the key players in prostate tumor cells, with short antisense oligodeoxynucleotides (ODNs) results in inhibition of prostate tumor growth. Particularly with regard to an application of these antisense drugs in vivo, we now investigated the usefulness of microbubble-enhanced ultrasound to deliver these ODNs into prostate cancer cells. Our short antisense AR ODNs were loaded onto the lipid surface of cationic gas-filled microbubbles by ion charge binding, and delivered into the cells by bursting the loaded microbubbles with ultrasound. In vitro experiments were initially performed to show that this kind of delivery system works in principle. In fact, transfection of prostate tumor cells with antisense AR ODNs using microbubble-enhanced ultrasound resulted in 49% transfected cells, associated with a decrease in AR expression compared to untreated controls. In vivo, uptake of a digoxigenin-labelled ODN was found in prostate tumour xenografts in nude mice following intratumoral or intravenous injection of loaded microbubbles and subsequent exposure of the tumour to ultrasound, respectively. Our results show that ultrasound seems to be the driving force of this delivery system. Uptake of the ODN was also observed in tumors after treatment with ultrasound alone, with only minor differences compared to the combined use of microbubbles and ultrasound.

Androgen Receptor Antagonists↗

PubMed2006

Deformable gas-filled microbubbles targeted to P-selectin.

Ultrasound contrast microbubbles have been successfully targeted to a number of intravascular disease markers. We hypothesized that targeted delivery could be improved further, by making the microbubbles deformable, leading to increased microbubble-endothelium adhesion contact area and stabilized adhesion. Activated leukocytes utilize such strategy; they deform after binding to inflamed endothelium in the vasculature. Lipid-shell microbubbles were targeted to the endothelial inflammatory protein P-selectin with a monoclonal anti-P-selectin antibody attached to the microbubble shell. Deformable microbubbles were created by controlled pressurization with partial gas loss, which generated an average excess shell surface area of approximately 30% and the formation of outward-projected wrinkles and folds. Targeted microbubble adhesion and deformability were assessed in the parallel plate flow chamber under shear flow. Sustained adhesion of deformable microbubbles at wall shear stresses between 0.4 and 1.35 dyn/cm(2) was consistently better than adhesion of wrinkle-free microbubbles. Over this shear range, targeted wrinkled microbubbles were deformed by shear flow, unlike wrinkle-free microbubbles. In a murine cremaster inflammation model, a significant improvement of deformable microbubble targeting was observed by intravital microscopy. Overall, the mechanical aspects of adhesion, such as particle shape, deformability and surface microstructure, are important in engineering efficient site-targeted particle-based agents for medical imaging and therapy.

Animals↗

PubMed2006

3D perfusion mapping in post-infarct mice using myocardial contrast echocardiography.

Myocardial contrast echocardiography (MCE) was used to construct three-dimensional maps of perfusion defects in closed-chest mice, with and without myocardial infarction (MI) induced by permanent coronary ligation. Contiguous, short-axis MCE cine images spanning the heart from apex to base were acquired at 1 mm elevations in each mouse. MCE images at each elevation were color-coded to indicate relative perfusion and were compared with postmortem histology. A strong correlation (R > 0.93) in the size of perfused areas was observed between in vivo measurements and the results of conventional ex vivo tissue staining. 3D multislice and 3D surface renderings of perfusion distribution were created and these perfusion maps also matched well with postmortem histology. These methods provide for the noninvasive determination of the total ischemic region placed at risk by coronary occlusion: this is a critical variable in assessing the potential of novel therapeutic agents to reduce MI size in murine models of ischemia/reperfusion injury.

Animals↗

PubMed2006

Targeting mucosal addressin cellular adhesion molecule (MAdCAM)-1 to noninvasively image experimental Crohn's disease.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is the second most common chronic inflammatory disorder worldwide; however, a noninvasive means of accurately assessing the severity and extent of intestinal inflammation is currently not available. The aim of the present study was to develop a noninvasive imaging modality to detect and evaluate ileitis in SAMP1/YitFc (SAMP) mice. METHODS: An image-enhancing ultrasound (US) contrast agent, consisting of encapsulated gaseous microbubbles (MB), was developed specifically to bind mucosal addressin cellular adhesion molecule-1 (MAdCAM-1), a mucosal-restricted addressin up-regulated during gut inflammation. MAdCAM-1-targeted MB (MB(M)) were tested for binding specificity on MAdCAM-1 protein and tumor necrosis factor (TNF)-stimulated SVEC4-10 endothelial cells using an in vitro flow chamber assay and for their ability to detect and quantify ileitis by intravital microscopy and transabdominal US. RESULTS: Under in vitro flow conditions, a 100-fold increase in MB(M) binding was observed on MAdCAM-1 protein compared with nonspecific MB (P < .001). TNF-stimulated endothelial cells bound significantly more MB(M) vs nonspecific MB (P < .001), which was abrogated after preincubation with anti-MAdCAM-1 antibodies (P < .001). In vivo, MB(M) specifically accumulated in focal areas of ileal inflammation and produced stronger acoustic echoes, measured by average video intensity, in SAMP vs uninflamed AKR mice (P < .001) or SAMP given nonspecific MB (P < .001). MB(M)-specific video intensity showed a strong positive correlation with total ileal inflammatory scores (R2 = 0.92). CONCLUSIONS: We have developed a novel intravascular US contrast agent targeting MAdCAM-1 that specifically detects and quantifies intestinal inflammation in experimental ileitis, providing the potential for a reliable, noninvasive means to diagnose and monitor disease in patients with IBD.

Animals↗

PubMed2006

Microbubble contrast agents: targeted ultrasound imaging and ultrasound-assisted drug-delivery applications.

The use of microbubble contrast agents for general tissue delineation and perfusion enjoys steady interest in ultrasound imaging. Microbubbles as contrast materials require a small dosage and show excellent detection sensitivity. Targeting ligands on the surface of microbubbles permit the selective accumulation of these particles in the areas of interest, which show an up-regulated level of receptor molecules on vascular endothelium. Selective contrast imaging of inflammation, ischemia-reperfusion injury, angiogenesis, and thrombosis has been achieved in animal models. Ultrasound-assisted drug delivery and activation, performed by combining microbubble agent containing drug substances or coadministered with pharmaceutical agents (including plasmid DNA for transfection), has been achieved in multiple model systems in vitro and in vivo. Ultrasound and microbubbles-based targeted acceleration of the thrombolytic enzyme action already have reached clinical trials. Overall, microbubble targeting and ultrasound-assisted microbubble-based drug-delivery systems will offer a step toward the application of targeted personalized diagnostics and therapy.

Animals↗

PubMed2006

Effect of microbubble ligation to cells on ultrasound signal enhancement: implications for targeted imaging.

OBJECTIVES: Molecular imaging with contrast-enhanced ultrasound (CEU) relies on the detection of microbubbles retained in regions of disease. The aim of this study was to determine whether microbubble attachment to cells influences their acoustic signal generation and stability. MATERIALS AND METHODS: Biotinylated microbubbles were attached to streptavidin-coated plates to derive density versus intensity relations during low- and high-power imaging. To assess damping from microbubble attachment to solid or cell surfaces, in vitro imaging was performed for microbubbles charge-coupled to methacrylate spheres and for vascular cell adhesion molecule-1-targeted microbubbles attached to endothelial cells. RESULTS: Signal enhancement on plates increased according to acoustic power and microbubble site density up to 300 mm. Microbubble signal was reduced by attachment to solid spheres during high- and low-power imaging but was minimally reduced by attachment to endothelial cells and only at low power. CONCLUSION: Attachment of targeted microbubbles to rigid surfaces results in damping and a reduction of their acoustic signal, which is not seen when microbubbles are attached to cells. A reliable concentration versus intensity relationship can be expected from microbubble attachment to 2-dimensional surfaces until a very high site density is reached.

Animals↗

PubMed2005

Assessment of endogenous and therapeutic arteriogenesis by contrast ultrasound molecular imaging of integrin expression.

BACKGROUND: We hypothesized that molecular imaging with contrast-enhanced ultrasound (CEU) and microbubbles targeted to endothelial integrins could be used to noninvasively assess early angiogenic responses to ischemia and growth factor therapy. METHODS AND RESULTS: Hindlimb ischemia was produced in 48 rats by ligation of an iliac artery. Half of the animals received intramuscular sustained-release fibroblast growth factor-2 (FGF-2). Immediately after ligation and at subsequent intervals from 4 to 28 days, blood flow and oxygen tension in the proximal adductor muscles were measured by CEU perfusion imaging and phosphor quenching, respectively. Targeted CEU imaging of alpha(v)- and alpha5beta1-integrin expression was performed with microbubbles bearing the disintegrin echistatin. Iliac artery ligation produced a 65% to 70% reduction in blood flow and oxygen tension. In untreated ischemic muscle, muscle flow and oxygen tension partially recovered by days 14 to 28. In these animals, signal from integrin-targeted microbubbles was intense and peaked before flow increase (days 4 to 7). In comparison to untreated animals, FGF-2-treated muscle had a greater rate and extent of blood flow recovery and greater signal intensity from integrin-targeted microbubbles, which peaked before maximal recovery of flow. On immunohistology, arteriolar but not capillary density increased in the ischemic limb after ligation, the rate and degree of which were greater in FGF-2-treated rats. Immunofluorescence demonstrated intense staining for alpha(v) in arterioles, the temporal course of which correlated with targeted imaging. CONCLUSIONS: Targeted CEU can be used to assess endogenous and therapeutic arteriogenesis before recovery of tissue perfusion. These results suggest that molecular imaging of integrin expression may be useful for evaluating proangiogenic therapies.

Animals↗

PubMed2005

Simultaneous integrin alphavbeta3 and glycoprotein IIb/IIIa inhibition causes reduction in infarct size in a model of acute coronary thrombosis and primary angioplasty.

OBJECTIVE: We tested the hypothesis that simultaneous inhibition of the endothelial integrin alpha(v)beta(3) and the platelet glycoprotein IIb/IIIa receptor will substantially reduce infarct size in a model of acute coronary thrombosis and primary angioplasty. METHODS: Dogs were subjected to thrombus formation in the left anterior descending coronary artery followed by primary angioplasty. Prior to angioplasty, they were randomized into 3 treatment groups. Group 1 (n=7) received saline; Group 2 (n=9) received MK-383 that inhibits only IIb/IIIa; and Group 3 (n=9) received CP-4715, that inhibits both IIb/IIIa and alpha(v)beta(3). RESULTS: There was a 59% reduction in infarct size in dogs receiving CP-4715 compared to controls (p=0.002) and a 37% reduction compared to the dogs receiving MK-383 (p=0.04). Myocardium microthrombi were seen to be reduced similarly with both drugs on post-mortem (99m)Tc-DMP444 autoradiography that reflects in vivo IIb/IIIa receptor activity. In vivo imaging using echistatin-conjugated and leukocyte-targeted microbubbles revealed significant alpha(v)beta(3) inhibition and reduction in active leukocyte recruitment only in Group 3 dogs. Myocardial blood flow and regional function after reperfusion were also significantly better in this group. CONCLUSION: Simultaneous inhibition of IIb/IIIa and alpha(v)beta(3) causes a marked reduction in infarct size in a model of acute coronary thrombosis and primary PTCA that is associated with reduced myocardial microthrombi and inflammation, as well as improved myocardial blood flow and regional function. These results may have important implications in the treatment of acute coronary syndromes.

Acute Disease↗

PubMed2005

Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumor-binding peptide arginine-arginine-leucine.

Endothelial cells (EC) of angiogenic tumor vasculature are characterized by altered expression of molecular markers on their surface. Numerous peptides have been identified that specifically bind tumor angiogenic endothelium, including the tripeptide arginine-arginine-leucine (RRL). We hypothesized that ultrasound contrast microbubbles (MB) targeted via linkage with RRL would specifically adhere to tumor angiogenic endothelium versus normal myocardium, and that this selective adhesion could be detected ultrasonically. Microbubbles were conjugated to cyclic peptides containing either RRL (RRL-MB) or a glycine control sequence (control-MB). As measured in a parallel plate flow chamber, in vitro adhesion of RRL-MBs was three times greater to cultured tumor-derived ECs than to normal ECs (P < 0.01), demonstrating selective binding of RRL-MBs to tumor endothelium. Mice bearing s.c. Clone C or PC3 tumors were given i.v. injections of fluorescent RRL to show in vivo localization to tumor vasculature or were ultrasonically imaged following i.v. injections of targeted contrast MBs. Ultrasound images showed strong RRL-MB contrast enhancement within the tumors but not the control tissue myocardium. Control-MBs caused minimal enhancement in either tissue. Quantitative acoustic videointensity was significantly greater for the tumors than the hearts (5 +/- 1 versus 0.5 +/- 1 intensity units; P = 0.001). These data show that ultrasound contrast MBs targeted to tumor vasculature via RRL preferentially adhere to tumor versus normal vasculature and that this selective adherence can be detected with ultrasound. Targeted microbubbles may thus offer a noninvasive contrast-enhanced ultrasound imaging technique for the functional imaging of tumor neovascularization, and may have further implications for therapeutic tumor targeting.

Animals↗

PubMed2005

Ultrasound-microbubble-induced neovascularization in mouse skeletal muscle.

Ultrasound-microbubble (US-MB) interactions stimulate neovascularization in rat gracilis muscle (GM). We examined microvascular remodeling (MVR) in GMs of C57BL/6 and balb/C mice following ultrasonic MB destruction. A range of MB dosages were administered IV, and exposed GMs received US. Muscles harvested 3, 7 and 14 d posttreatment were stained for vascular markers and assessed for changes in microvessel number, diameter and length. Muscles receiving a low MB dose (LMBD) and US showed significant increases in microvascular density after 3 d, returning to sham levels after one week. A MB dose producing maximum capillary disruptions was then established. This high MB dose (HMBD) facilitated significant MVR in C57BL/6 mice after one week. Balb/C GMs exhibited neovascularization 3 d, but not 7 or 14 d, following US-HMBD treatment. We conclude that HMBD in C57BL/6 mice induces a more sustained neovascularization response compared to balb/C or LMBD-treated C57BL/6 muscles; however, this response is still impermanent.

Animals↗

PubMed2005

Acoustic radiation force enhances targeted delivery of ultrasound contrast microbubbles: in vitro verification.

Recent research has shown that targeted ultrasound contrast microbubbles achieve specific adhesion to regions of intravascular pathology, but not in areas of high flow. It has been suggested that acoustic radiation can be used to force free-stream microbubbles toward the target, but this has not been verified for actual targeted contrast agents. We present evidence that acoustic radiation indeed increases the specific targeted accumulation of microbubbles. Lipid microbubbles bearing an antibody as a targeting ligand were infused through a microcapillary flow chamber coated with P-selectin as the target protein. A 2.0 MHz ultrasonic pulse was applied perpendicular to the flow direction. Microbubble accumulation was observed on the flow chamber surface opposite the transducer. An acoustic pressure of 122 kPa enhanced microbubble adhesion up to 60-fold in a microbubble concentration range of 0.25 x 10(6) to 75 x 106) ml(-1). Acoustic pressure mediated the greatest adhesion enhancement at concentrations within the clinical dosing range. Acoustic pressure enhanced targeting nearly 80-fold at a wall shear rate of 1244 s(-1), suggesting that this mechanism is appropriate for achieving targeted microbubble delivery in high-flow vessels. Microbubble adhesion increased with the square of acoustic pressure between 25 and 122 kPa, and decreased substantially at higher pressures.

Antibodies↗

PubMed2004

Microvascular remodeling and accelerated hyperemia blood flow restoration in arterially occluded skeletal muscle exposed to ultrasonic microbubble destruction.

We showed previously that microbubble destruction with pulsed 1-MHz ultrasound creates a bioeffect that stimulates arteriogenesis and a chronic increase in hyperemia blood flow in normal rat muscle. Here we tested whether ultrasonic microbubble destruction can be used to create a microvascular remodeling response that restores hyperemia blood flow to rat skeletal muscle affected by arterial occlusion. Pulsed ultrasound (1 MHz) was applied to gracilis muscles in which the lateral feed artery was occluded but the medial feed artery was left intact. Control muscles were similarly occluded but did not receive ultrasound, microbubbles, or both. Hyperemia blood flow and number of smooth muscle (SM) alpha-actin-positive vessels, >30-mum arterioles, and capillaries per fiber were determined 7, 14, and 28 days after treatment. In ultrasound-microbubble-treated muscles, lateral region hyperemia blood flow was increased at all time points and restored to normal at day 28. The number of SM alpha-actin vessels per fiber was increased over control in this region at days 7 and 14 but decreased by day 28, when larger-diameter arterioles became more prevalent in the medial region. The number of capillaries per fiber was increased over control only at day 7 in the lateral region and only at days 7 and 14 in the medial region, indicating that the angiogenesis response was transient and likely did not contribute significantly to flow restoration at day 28. We conclude that ultrasonic microbubble destruction can be tailored to stimulate an arteriogenesis response that restores hyperemia blood flow to skeletal muscle in a rat model of arterial occlusion.

Animals↗

PubMed2004

Binding and detachment dynamics of microbubbles targeted to P-selectin under controlled shear flow.

This study was performed to assess the binding kinetics of a targeted microbubble contrast agent exposed to shear stress. An ultrasound contrast targeted to P-selectin was designed by conjugating monoclonal antibodies against murine P-selectin (RB40.34) to the lipid monolayer shell of the microbubble using poly(ethylene glycol)-biotin-streptavidin. The attachment and detachment of targeted microbubbles to P-selectin immobilized on a culture dish were assessed in a parallel-plate flow chamber. Targeted microbubbles (5 x 10(6) particles/ml) drawn through the flow chamber coated with P-selectin (109 sites/microm(2)) at a shear stress of 0.3 dyn/cm(2) accumulated at a rate of 565 mm(-2) min(-1). Attachment rates increased at higher plate surface densities of P-selectin, and microbubble detachment was reduced. Accumulation rate first increased with shear stress, reached a maximum at approximately 0.6 dyn/cm(2) and then decreased. Control experiments on a plate that lacked P-selectin, or was blocked with mAb RB40.34, resulted in minimal bubble attachment. Microbubble detachment was tested by ramping up shear stress at 30-s intervals. Half-maximal detachment was reached at 34 dyn/cm(2). Overall, accumulation and retention of targeted ultrasound contrast agents is possible under physiologic flow conditions and is strongly influenced by shear stress and surface density of the target receptor.

Antibodies, Monoclonal↗

PubMed2004

Detection of individual microbubbles of ultrasound contrast agents: imaging of free-floating and targeted bubbles.

RATIONALE AND OBJECTIVES: During echo examinations with microbubble contrast, individual "dots" of ultrasound reflection can be visualized. To address the question whether these signals represent individual microbubbles, very dilute suspensions of ultrasound contrast agents or individual microbubbles attached to Petri dishes were prepared and studied by ultrasound imaging. METHODS: Microbubble suspensions were diluted in saline and evaluated by a clinical ultrasound imaging system. Microbubble concentration was verified by Coulter counter. Single microbubble preparation on a Petri dish was established by streptavidin-biotin interaction under microscopy control and subjected to ultrasound imaging. RESULTS: Ultrasound of dilute microbubble dispersions demonstrated distinct white foci; concentration of these sites was consistent with signals from individual microbubbles as determined by Coulter. Individual microbubbles immobilized on polystyrene were also visualized by ultrasound. CONCLUSION: Ultrasound medical systems can resolve backscatter signals from individual microbubbles of ultrasound contrast, both in solution and in the targeted immobilized state, implying picogram sensitivity.

Contrast Media↗

PubMed2004

Microvascular recruitment is an early insulin effect that regulates skeletal muscle glucose uptake in vivo.

Insulin increases glucose disposal into muscle. In addition, in vivo insulin elicits distinct nitric oxide synthase-dependent vascular responses to increase total skeletal muscle blood flow and to recruit muscle capillaries (by relaxing resistance and terminal arterioles, respectively). In the current study, we compared the temporal sequence of vascular and metabolic responses to a 30-min physiological infusion of insulin (3 mU. min(-1). kg(-1), euglycemic clamp) or saline in rat skeletal muscle in vivo. We used contrast-enhanced ultrasound to continuously quantify microvascular volume. Insulin recruited microvasculature within 5-10 min (P < 0.05), and this preceded both activation of insulin-signaling pathways and increases in glucose disposal in muscle, as well as changes in total leg blood flow. Moreover, l-NAME (N(omega)-nitro-l-arginine-methyl ester), a specific inhibitor of nitric oxide synthase, blocked this early microvascular recruitment (P < 0.05) and at least partially inhibited early increases in muscle glucose uptake (P < 0.05). We conclude that insulin rapidly recruits skeletal muscle capillaries in vivo by a nitric oxide-dependent action, and the increase in capillary recruitment may contribute to the subsequent glucose uptake.

Animals↗

PubMed2003

Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1.

BACKGROUND: Noninvasive techniques for detecting acute cardiac transplant rejection are limited. We hypothesized that ultrasound contrast microbubbles targeted to the endothelial cell (EC) inflammatory marker intercellular adhesion molecule-1 (ICAM-1) would selectively bind to rejecting versus nonrejecting myocardium and that myocardial contrast echocardiography can therefore detect acute rejection. METHODS AND RESULTS: Lipid-based microbubbles were conjugated to anti-rat ICAM-1 (MBICAM) or isotype control antibody (MBControl). In vitro MBICAM adhesion to cultured rat ECs, as assessed in a parallel plate flow apparatus, was greater to inflammatory versus normal ECs (11+/-3 versus 3+/-2 microbubbles/EC, P<0.005). In vivo abdominal heterotopic heart transplantation was performed in rats (rejection group: Brown Norway to Lewis strain; control group: Lewis to Lewis or Brown Norway to Brown Norway). Triggered myocardial contrast echocardiography was performed during intravenous MBICAM or MBControl (2.5x10(6)) injection on postoperative day 5. Myocardial videointensity from adhered MBICAM was significantly higher in rejecting (n=8) versus control (n=7) rats (10+/-4 versus 1+/-4 U, P=0.01). Postmortem histology showed normal myocardium in control rats, whereas allograft myocardium demonstrated grade III to IV rejection and strong immunohistochemical ICAM-1 staining. CONCLUSIONS: Preferential adherence of ICAM-1-targeted microbubbles to rejecting versus nonrejecting rat cardiac transplant myocardium can be detected ultrasonically. Targeted microbubbles may thus offer a noninvasive ultrasound imaging technique for the detection of acute cardiac transplant rejection and other processes characterized by endothelial dysfunction.

Acute Disease↗

PubMed2003

Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha(v)beta3.

BACKGROUND: Angiogenesis is a critical determinant of tumor growth and metastasis. We hypothesized that contrast-enhanced ultrasound (CEU) with microbubbles targeted to alpha(v)-integrins expressed on the neovascular endothelium could be used to image angiogenesis. METHODS AND RESULTS: Malignant gliomas were produced in 14 athymic rats by intracerebral implantation of U87MG human glioma cells. On day 14 or day 28 after implantation, CEU was performed with microbubbles targeted to alpha(v)beta3 by surface conjugation of echistatin. CEU perfusion imaging with nontargeted microbubbles was used to derive tumor microvascular blood volume and blood velocity. Vascular alpha(v)-integrin expression was assessed by immunohistochemistry, and microbubble adhesion was characterized by confocal microscopy. Mean tumor size increased markedly from 14 to 28 days (2+/-1 versus 35+/-14 mm2, P<0.001). Tumor blood volume increased by approximately 35% from day 14 to day 28, whereas microvascular blood velocity decreased, especially at the central portions of the tumors. On confocal microscopy, alpha(v)beta3-targeted but not control microbubbles were retained preferentially within the tumor microcirculation. CEU signal from alpha(v)beta3-targeted microbubbles in tumors increased significantly from 14 to 28 days (1.7+/-0.4 versus 3.3+/-1.0 relative units, P<0.05). CEU signal from alpha(v)beta3-targeted microbubbles was greatest at the periphery of tumors, where alpha(v)-integrin expression was most prominent, and correlated well with tumor microvascular blood volume (r=0.86). CONCLUSIONS: CEU with microbubbles targeted to alpha(v)beta3 can noninvasively detect early tumor angiogenesis. This technique, when coupled with changes in blood volume and velocity, may provide insights into the biology of tumor angiogenesis and be used for diagnostic applications.

Animals↗