Clinical benefit of switching from paclitaxel to docetaxel or vice versa in cutaneous angiosarcoma patients resistant to first taxane chemotherapy.

Cutaneous angiosarcoma (CAS) is a rare soft-tissue sarcoma of vascular endothelial origin. Paclitaxel (PTX) and docetaxel (DTX) are used as systemic chemotherapy; however, chemoresistance often occurs in CAS. Switching one taxane to the other (i.e., PTX to DTX, or vice versa) is an option when the first taxane is no longer effective in malignant cancers such as ovarian or breast cancer. However, the efficacy of the same strategy in CAS has not been reported. Herein, we report the clinical response of switching one taxane-based chemotherapy to the other in CAS patients with resistance to the first taxane. Twelve CAS patients were included for analyses. In all patients, the median overall survival from the start of the first taxane treatment was 29.0 months (range, 6.47-58.5). During the first taxane, the median PFS for all patients was 5.96 months (1.81-47.1). Similarly, the median (range) PFS for all patients during the second taxane was 5.87 months (1.60-18.2). Furthermore, the median OS was 22.7 months (PTX to DTX) and 39.5 months (DTX to PTX) (p = 0.307). The median PFS during the first taxane was 5.14 (PTX to DTX) and 12.5 months (DTX to PTX), respectively (p = 0.380). The median PFS during the second taxane was 3.5 (PTX to DTX) and 7.1 months (DTX to PTX), respectively (p = 0.906). The objective response rate, defined as the sum of complete response (CR) and partial response (PR) rates, was 16.7%. The disease control rate, defined as the sum of CR, PR, and stable disease rates, was 50%. The frequency of adverse events during the second taxane was the same between the two groups (p > 0.999). Our report suggests that CAS patients could benefit from the second taxane treatment if the tumor is resistant to the first taxane.

A case of allergic bronchopulmonary mycosis due to Schizophyllum commune with elevated serum carcinoembryonic antigen levels.

A 78-year-old Japanese woman without any history of asthma or smoking presented prolonged cough. Laboratory data showed elevated serum CEA levels and a chest CT revealed a mass with abnormal uptake in the left lower lobe. One month later, the mass spontaneously regressed, and CEA levels improved. However, the symptoms progressed during the observation period without treatment. Chest radiograph findings revealed collapse of the right middle lobe, and Schizophyllum commune was isolated from the mucous plugs; the patient was diagnosed with allergic bronchopulmonary mycosis (ABPM). Herein, we report the first case of ABPM caused by S. commune with elevated CEA levels and mimicking lung cancer.

GFAP-Negative Subcutaneous Sacrococcygeal Extraspinal Ependymoma.

Ependymomas are slowly growing glial tumors derived from the ependymal cells and usually occur in the central nervous system (CNS). Ependymomas rarely occur outside of the CNS and they are called extraspinal ependymomas. In spite of their metastatic potential, extraspinal ependymomas can be misdiagnosed for other benign mass like pilonidal cysts. The diagnosis is confirmed by histopathology and most of the cases are known to show glial fibrillary acidic protein (GFAP), S-100 protein, and keratin (AE1AE3) immunoreactivity. Herein, we present a case of GFAP-negative ependymoma, which presented as asymptomatic subcutaneous tumor of the left buttock and was clinically misdiagnosed as epidermal cyst. Our case indicates that ependymomas cannot be ruled out by lack of GFAP immunoreactivity and an asymptomatic subcutaneous mass could be a malignant tumor like ependymomas, which requires careful examinations.

DNA damage response induced by Etoposide promotes steroidogenesis via GADD45A in cultured adrenal cells.

Glucocorticoid production is regulated by adrenocorticotropic hormone (ACTH) via the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway in the adrenal cortex, but the changes in steroidogenesis associated with aging are unknown. In this study, we show that cell-autonomous steroidogenesis is induced by non-ACTH- mediated genotoxic stress in human adrenocortical H295R cells. Low-dose etoposide (EP) was used to induce DNA damage as a genotoxic stress, leading to cellular senescence. We found that steroidogenesis was promoted in cells stained with γH2AX, a marker of DNA damaged cells. Among stress-associated and p53-inducible genes, the expression of GADD45A and steroidogenesis-related genes was significantly upregulated. Immunofluorescence analysis revealed that GADD45A accumulated in the nuclei. Metabolite assay using cultured media showed that EP-treated cells were induced to produce and secrete considerable amounts of glucocorticoid. Knockdown of GADD45A using small interfering RNA markedly inhibited the EP-induced upregulation of steroidogenesis-related gene expression, and glucocorticoid production. A p38MAPK inhibitor, but not a PKA inhibitor, suppressed EP-stimulated steroidogenesis. These results suggest that DNA damage itself promotes steroidogenesis via one or more unprecedented non-ACTH-mediated pathway. Specifically, GADD45A plays a crucial role in the steroidogenic processes triggered by EP-stimulated genotoxic stress. Our study sheds new light on an alternate mechanism of steroidogenesis in the adrenal cortex.

An Excess of CYP24A1, Lack of CaSR, and a Novel lncRNA Near the PTH Gene Characterize an Ectopic PTH-Producing Tumor.

Thus far, only 23 cases of the ectopic production of parathyroid hormone (PTH) have been reported. We have characterized the genome-wide transcription profile of an ectopic PTH-producing tumor originating from a retroperitoneal histiocytoma. We found that the calcium-sensing receptor (CaSR) was barely expressed in the tumor. Lack of CaSR, a crucial braking apparatus in the presence of both intraparathyroid and, probably, serendipitous PTH expression, might contribute strongly to the establishment and maintenance of the ectopic transcriptional activation of the PTH gene in nonparathyroid cells. Along with candidate drivers with a crucial frameshift mutation or copy number variation at specific chromosomal areas obtained from whole exome sequencing, we identified robust tumor-specific cytochrome P450 family 24 subfamily A member 1 (CYP24A1) overproduction, which was not observed in other non-PTH-expressing retroperitoneal histiocytoma and parathyroid adenoma samples. We then found a 2.5-kb noncoding RNA in the PTH 3'-downstream region that was exclusively present in the parathyroid adenoma and our tumor. Such a co-occurrence might act as another driver of ectopic PTH-producing tumorigenesis; both might release the control of PTH gene expression by shutting down the other branches of the safety system (e.g., CaSR and the vitamin D3-vitamin D receptor axis).

Tumour hypoxia promotes melanoma growth and metastasis via High Mobility Group Box-1 and M2-like macrophages.

Hypoxia is a hallmark of cancer that is strongly associated with invasion, metastasis, resistance to therapy and poor clinical outcome. Tumour hypoxia affects immune responses and promotes the accumulation of macrophages in the tumour microenvironment. However, the signals linking tumour hypoxia to tumour-associated macrophage recruitment and tumour promotion are incompletely understood. Here we show that the damage-associated molecular pattern High-Mobility Group Box 1 protein (HMGB1) is released by melanoma tumour cells as a consequence of hypoxia and promotes M2-like tumour-associated macrophage accumulation and an IL-10 rich milieu within the tumour. Furthermore, we demonstrate that HMGB1 drives IL-10 production in M2-like macrophages by selectively signalling through the Receptor for Advanced Glycation End products (RAGE). Finally, we show that HMGB1 has an important role in murine B16 melanoma growth and metastasis, whereas in humans its serum concentration is significantly increased in metastatic melanoma. Collectively, our findings identify a mechanism by which hypoxia affects tumour growth and metastasis in melanoma and depict HMGB1 as a potential therapeutic target.

Regulatory T Cells in Melanoma Revisited by a Computational Clustering of FOXP3+ T Cell Subpopulations.

CD4(+) T cells that express the transcription factor FOXP3 (FOXP3(+) T cells) are commonly regarded as immunosuppressive regulatory T cells (Tregs). FOXP3(+) T cells are reported to be increased in tumor-bearing patients or animals and are considered to suppress antitumor immunity, but the evidence is often contradictory. In addition, accumulating evidence indicates that FOXP3 is induced by antigenic stimulation and that some non-Treg FOXP3(+) T cells, especially memory-phenotype FOXP3(low) cells, produce proinflammatory cytokines. Accordingly, the subclassification of FOXP3(+) T cells is fundamental for revealing the significance of FOXP3(+) T cells in tumor immunity, but the arbitrariness and complexity of manual gating have complicated the issue. In this article, we report a computational method to automatically identify and classify FOXP3(+) T cells into subsets using clustering algorithms. By analyzing flow cytometric data of melanoma patients, the proposed method showed that the FOXP3(+) subpopulation that had relatively high FOXP3, CD45RO, and CD25 expressions was increased in melanoma patients, whereas manual gating did not produce significant results on the FOXP3(+) subpopulations. Interestingly, the computationally identified FOXP3(+) subpopulation included not only classical FOXP3(high) Tregs, but also memory-phenotype FOXP3(low) cells by manual gating. Furthermore, the proposed method successfully analyzed an independent data set, showing that the same FOXP3(+) subpopulation was increased in melanoma patients, validating the method. Collectively, the proposed method successfully captured an important feature of melanoma without relying on the existing criteria of FOXP3(+) T cells, revealing a hidden association between the T cell profile and melanoma, and providing new insights into FOXP3(+) T cells and Tregs.

Syringocystadenocarcinoma Papilliferum in the Perianal Area.

Syringocystadenocarcinoma papilliferum (SCACP) is a very rare cutaneous adnexal neoplasm. SCACP presents histologic variability, and it is difficult to establish the diagnosis from a punch biopsy. SCACP has an overall configuration similar to that of syringocystadenoma papilliferum (SCAP). When we diagnose SCACP, the histologic features of SCAP can be contributing and immunohistochemical staining is useful. Our case shows the histologic variability of SCACP and the pitfalls of a punch biopsy for the diagnosis of SCACP.

Marked cortisol production by intracrine ACTH in GIP-treated cultured adrenal cells in which the GIP receptor was exogenously introduced.

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing's syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (-) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3ß2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.

CD8⁺ tumor-infiltrating lymphocytes at primary sites as a possible prognostic factor of cutaneous angiosarcoma.

Tumor-infiltrating lymphocytes (TILs) have been reported as a prognostic factor in various cancers and are a promising target for immunotherapy. To investigate whether TILs have any impact on the prognosis of angiosarcoma patients, 55 non-treated patients (40 patients at stage 1 with cutaneous localized tumors, 4 patients at stage 2 with lymph node metastases and 11 patients at stage 3 with distant metastases) with angiosarcoma were evaluated retrospectively by immunohistochemistry stained CD4, CD8, FOXP3 and Ki67. The Kaplan-Meier method was used to estimate overall survival with patients at stage 1. Survival differences were analyzed by the log-rank test. Patients with higher numbers of CD8(+) TILs in their primary tumors survived significantly longer compared with patients with lower values. Moreover, the number of CD8 in TILs was positively correlated with a distant metastasis-free period. The total number of primary TILs (CD4 plus CD8) and CD8(+) primary TILs of stage 3 patients with distant metastases was positively correlated with their overall survival. To evaluate whether CD8(+) effector T cells are activated or differentiated, flow cytometric analysis of peripheral blood mononuclear cells (PBMC) was performed. The percentages of CD8(+) T cells producing IFN-γ in PBMC were significantly higher in patients with angiosarcoma (n = 10) compared not only with that of healthy controls (n = 20) but also patients with advanced melanoma (n = 11). These results suggest that anti-tumor immunity is clinically relevant in angiosarcoma.

Optimization of Ultrasound-mediated Anti-angiogenic Cancer Gene Therapy.

Ultrasound-targeted microbubble destruction (UTMD) can be used to deliver silencing gene therapy to tumors. We hypothesized that UTMD would be effective in suppressing angiogenesis within tumors, and that modulation of the ultrasound pulsing intervals (PI) during UTMD would affect the magnitude of target knockdown. We performed UTMD of vascular endothelial growth factor receptor-2 (VEGFR2) short hairpin (sh)RNA plasmid in an heterotopic mammary adenocarcinoma model in rats, evaluating PIs of 2, 5, 10, and 20 seconds. We demonstrated that UTMD with a PI of 10 seconds resulted in the greatest knockdown of VEGFR2 by PCR, immunostaining, western blotting, smaller tumor volumes and perfused areas, and lower tumor microvascular blood volume (MBV) and flow by contrast-enhanced ultrasound (CEU) compared with UTMD-treated tumors at 2, 5, and 20 seconds, control tumors, tumors treated with intravenous shRNA plasmid and scrambled plasmid. CEU perfusion assessment using the therapeutic probe demonstrated that tumors were fully replenished with microbubbles within 10 seconds, but incompletely replenished at PI-2 and PI-5 seconds. In conclusion, for anti-VEGFR2 cancer gene therapy by UTMD, PI of 10 seconds results in higher target knockdown and a greater anti-angiogenic effect. Complete replenishment of tumor vasculature with silencing gene-bearing microbubbles in between destructive pulses of UTMD is required to maximize the efficacy of anti-angiogenic cancer gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e94; doi:10.1038/mtna.2013.20; published online 21 May 2013.

Statin therapy prevents expansive remodeling in venous bypass grafts.

BACKGROUND: Venous grafts (VG) have high failure rates by 10 years in aortocoronary bypass surgery. We have previously shown that expansive remodeling followed by increased LDL retention are early atherosclerotic changes in experimental VG placed in the arterial circulation. The objective of this study was to determine whether statin therapy prevents these expansive remodeling changes. METHODS AND RESULTS: Reversed jugular vein-to-common carotid artery interposition graft was constructed in 27 cholesterol-fed (0.5%) rabbits. Rabbits were randomized either to control or atorvastatin (5 mg/kg/day) groups, starting two weeks prior to vein graft implantation and continuing until sacrifice at 1 or 12 weeks post-surgery. Ultrasound measurements of arterial luminal cross-sectional area (CSA) were done at day 3 and at 4, 8 and 12 weeks post-surgery. Histomorphometric measurements were performed following sacrifice at 12 weeks. Atorvastatin treatment significantly decreased total plasma cholesterol levels at 4, 8 and 12 weeks (12 weeks: 6.7 ± 4.2 mmol/L versus control 38.7 ± 10.6 mmol/L, p<0.0002). Atorvastatin significantly reduced expansive remodeling at 4, 8 and 12 weeks (lumen CSA: 44.6 ± 6.6 mm(2) versus control 77.6 ± 10.7 mm(2), p<0.0001). Intimal CSA by histomorphometry was also significantly reduced by atorvastatin at 12 weeks (5.59 ± 2.19 mm(2) versus control 9.57 ± 2.43 mm(2), p<0.01). VG macrophage infiltration, MMP-2 activity and metalloelastase activity were reduced in the atorvastatin treated group. CONCLUSION: Atorvastatin inhibits both expansive remodeling and intimal hyperplasia in arterialized VG, likely through inhibition of macrophage infiltration and reduction of tissue proteolytic activity. The mechanism proposed above may be important for preventing VG atherosclerosis and late VG failure.

Impaired glucose metabolism in Japanese patients with acromegaly is restored after successful pituitary surgery if pancreatic {beta}-cell function is preserved.

OBJECTIVE: Impaired glucose metabolism is common in acromegaly, but it is not clear how glucose metabolism is impaired or what predicts its restoration after cure of the disease. To identify factors involved in the impairment of glucose metabolism in acromegaly, we evaluated clinical parameters before and immediately after surgical cure of the disease. DESIGN AND METHODS: We retrospectively analyzed clinical data of 92 consecutive Japanese patients with acromegaly who underwent successful pituitary surgery. Patients who had received medical therapy for acromegaly or insulin treatment for diabetes were excluded. We evaluated insulin resistance (IR) and pancreatic ß-cell function in addition to GH and IGF1 levels before and after surgery. Results In this study of Japanese patients with acromegaly, average body mass index (BMI) was 23.4, and no patient had a BMI>30. IR was involved in the impairment of glucose metabolism, which was restored upon surgical cure of acromegaly if ß-cell function was preserved. Insufficient ß-cell function did not improve after normalization of GH/IGF1 and was associated with impaired glucose metabolism before and after surgery. RESULTS: of receiver operating characteristic analysis of preoperative clinical parameters suggest that insulinogenic index (IGI) >0.50 best predicts restoration of normal glucose metabolism upon cure of acromegaly in Japanese patients. CONCLUSIONS: IR impairs glucose metabolism in acromegaly. Once ß-cell function is impaired, abnormal glucose metabolism persists even after cure of acromegaly. IGI>0.50 indicates that ß-cell function is preserved in non-obese Japanese patients with acromegaly.

Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart via ultrasound targeted microbubble destruction enhances cardiac repair.

AIMS: Ultrasound-targeted microbubble destruction (UTMD) uses ultrasound energy to selectively deliver genes into the myocardium using plasmids conjugated to microbubbles. We hypothesized that repeated delivery of stem cell-mobilizing genes could boost the ability of this therapy to enhance cardiac repair and ventricular function after a myocardial infarction. METHODS AND RESULTS: Beginning 7 days after coronary artery ligation, stem cell factor (SCF) and stromal cell-derived factor (SDF)-1α genes were administered to adult rats using 1, 3, or 6 UTMD treatments (repeat 1, 3, and 6 groups) at 2-day intervals (control=6 treatments with empty plasmid). Cardiac function (echocardiography) and myocardial perfusion (myocardial contrast echocardiography) were assessed on Days -7, 0, and 24 relative to the first treatment. Histological and biochemical assessments were performed on Day 24. Multiple UTMD treatments were associated with an increased presence of myocardial SCF and SDF-1α proteins and their receptors (vs. control and Repeat 1). All UTMD recipients exhibited increased vascular densities and smaller infarct regions (vs. control), with the highest ventricular densities in response to multiple treatments. Myocardial perfusion and ventricular function at Day 24 also improved progressively (vs. control) with the number of UTMD treatments. CONCLUSIONS: Targeted ultrasound delivery of SCF and SDF-1α genes to the infarcted myocardium recruited progenitor cells and increased vascular density. Multiple UTMD treatments enhanced tissue repair, perfusion, and cardiac function. Repeated UTMD therapy may be applied to tailor the number of interventions required to optimize cardiac regeneration after an infarction.

Olfactory ensheathing cell tumor: case report.

Subfrontal schwannomas, sometimes referred to as olfactory groove schwannomas, are rare tumors (34 cases reported to date). Despite the name and several theories proposed in the literature, there is no officially recognized description of the tumor's cell origin. Yasuda proposed the concept of an olfactory ensheathing cell (OEC) tumor in 2006. Olfactory ensheathing cells are glial cells that ensheath the axons of the first cranial nerve. Microscopically, both olfactory ensheathing cells and Schwann cells have similar morphological and immunohistochemical features. However, immunohistochemically olfactory ensheathing cells are negative for Leu7 and Schwann cells positive. A 30-year-old woman presented with a subfrontal, extraaxial, enhancing tumor, and underwent gross total resection. Immunohistochemical reactivity data suggested a schwannoma (positive for S-100 and negative for epithelial membrane antigen). However, the tumor was negative for Leu7. Accordingly, our final diagnosis was that of an OEC tumor. Subfrontal schwannoma immunohistochemical staining, if negative for Leu7, is indicative of an OEC tumor. It is possible that schwannoma-like extraaxial tumors at the anterior skull base are OEC tumors, which negative Leu7 staining can confirm.

Evaluation of the chemosensitivity of primary cultured malignant melanoma cells using the collagen gel droplet-embedded culture drug sensitivity test.

Malignant melanoma usually shows resistance to a standard chemotherapy regimen. A useful in vitro method to evaluate individual chemosensitivity is required to select effective anti-cancer drugs. This study aimed to establish in vitro tumor response testing for malignant melanoma. We determined the chemosensitivity of primary cultured melanoma cells using the collagen gel droplet-embedded culture drug sensitivity test (CD-DST). Nineteen tests were carried out for 15 cases of malignant melanoma. Primary cultured melanoma cells in collagen gel droplets were exposed to anti-cancer drugs, including cisplatin, adriamycin, dacarbazine, nimustine and vincristine. After a 7-day incubation in a serum-free medium, living melanoma cells in a collagen droplet were detected by image analysis after staining with Neutral red reagent. In vitro drug exposure conditions were determined to reproduce the value of the plasma area under the time-drug concentration curve in vivo. The rate of evaluation of the primary culture of melanoma cells was 78.9% (15/19 tests). The chemosensitivity of cisplatin, adriamycin, dacarbazine, nimustine and vincristine was 15, 62, 0, 0 and 62%, respectively. Dacarbazine was not suitable for CD-DST due to its prodrug characteristics. The CD-DST method was able to evaluate the chemosensitivity of malignant melanoma to anti-cancer drugs in vitro. This method can also be applied to estimate the efficacy of newly developed anti-cancer drugs in vitro.

Ultrasound-targeted gene delivery induces angiogenesis after a myocardial infarction in mice.

OBJECTIVES: This study evaluated the capacity of ultrasound-targeted microbubble destruction (UTMD) to deliver angiogenic genes, improve perfusion, and recruit progenitor cells after a myocardial infarction (MI) in mice. BACKGROUND: Angiogenic gene therapy after an MI may become a clinically relevant approach to improve the engraftment of implanted cells if targeted delivery can be accomplished noninvasively. The UTMD technique uses myocardial contrast echocardiography to target plasmid gene delivery to the myocardium and features low toxicity, limited immunogenicity, and the potential for repeated application. METHODS: Empty plasmids (control group) or those containing genes for vascular endothelial growth factor (VEGF), stem cell factor (SCF), or green fluorescent protein (to visualize gene delivery) were incubated with perflutren lipid microbubbles. The microbubble-deoxyribonucleic acid mixture was injected intravenously into C57BL/6 mice at 7 days after coronary artery ligation (MI). The UTMD technique facilitated transgene release into the myocardium. Twenty-one days after MI, myocardial perfusion and function were assessed by contrast echocardiography. Protein expression was quantified by Western blot and enzyme-linked immunosorbent assay. Flow cytometry quantified progenitor cell recruitment to the heart. Blood vessel density was evaluated immunohistochemically. RESULTS: Green fluorescent protein expression in the infarcted myocardium demonstrated gene delivery. Myocardial VEGF and SCF levels increased significantly in the respective groups (p < 0.05). The physiologic impact of VEGF and SCF gene delivery was confirmed by increased myocardial recruitment of VEGF receptor 2- and SCF receptor (c-kit)-expressing cells, respectively (p < 0.05). Consequently, capillary and arteriolar density (Factor VIII and alpha-smooth muscle actin staining), myocardial perfusion, and cardiac function were all enhanced (p < 0.01 relative to control group) in recipients of VEGF or SCF. CONCLUSIONS: Noninvasive UTMD successfully delivered VEGF and SCF genes into the infarcted heart, increased vascular density, and improved myocardial perfusion and ventricular function. The UTMD technique may be an ideal method for noninvasive, repeated gene delivery after an MI.

Molecular imaging of endothelial progenitor cell engraftment using contrast-enhanced ultrasound and targeted microbubbles.

AIMS: Imaging methods to track the fate of progenitor cells after their delivery would be useful in assessing the efficacy of cell-based therapies. We hypothesized that contrast-enhanced ultrasound (CEU) using microbubbles targeted to a genetically engineered cell-surface marker on endothelial progenitor cells (EPCs) would allow the targeted imaging of vascular engraftment. METHODS AND RESULTS: Rodent bone marrow-derived EPCs were isolated, cultured, and transfected to express the marker protein, H-2Kk, on the cell surface. Non-transfected EPCs and EPCs transfected with either null plasmid or Firefly luciferase served as controls. Control microbubbles (MB(C)) and microbubbles targeted to H-2Kk expressed on EPCs (MB(H-2Kk)) were constructed. Binding of targeted microbubbles to EPCs was assessed in vitro using a parallel plate flow chamber system. CEU imaging of EPC-targeted microbubbles was assessed in vivo using subcutaneously implanted EPC-supplemented Matrigel plugs in rats. In flow chamber experiments, there was minimal attachment of microbubbles to plated control EPCs. Although numbers of adhered MB(C) were also low, there was greater and more diffuse attachment of MB(H-2Kk) to plated H-2Kk-transfected EPCs. Targeted CEU demonstrated marked contrast enhancement at the periphery of the H-2Kk-transfected EPC-supplemented Matrigel plug for MB(H-2Kk,) whereas contrast enhancement was low for MB(C). Contrast enhancement was also low for both microbubbles within control mock-transfected EPC plugs. The signal intensity within the H-2Kk-transfected EPC plug was significantly greater for MB(H-2Kk) when compared with MB(C). CONCLUSION: Microbubbles targeted to a genetically engineered cell-surface marker on EPCs exhibit specific binding to EPCs in vitro. These targeted microbubbles bind to engrafted EPCs in vivo within Matrigel plugs and can be detected by their enhancement on CEU imaging.