Michael Dobryansky

Expression of von Willebrand factor, an endothelial cell marker, is up- regulated by angiogenesis factors: A potential method for objective assessment of tumor angiogenesis

von Willebrand factor (vWF), a glycoprotein produced uniquely by endothelial cells and megakaryocytes, is routinely used to identify vessels in tissue sections. Vessel density in tumor specimens, as determined by immuno‐histochemical staining for vWF or other endothelial cell markers, is a negative prognostic factor for many solid tumors. vWF is heterogeneously distributed throughout the vasculature, transcriptional control in response to the tissue microenvironment being responsible for local variations in endothelial cell levels of vWF. Here, we report that fibroblast growth factor‐2 and vascular endothelial growth factor, potent angiogenesis inducers expressed in a variety of tumors, up‐regulate expression of vWF mRNA and protein in cultured endothelial cells with a synergistic effect. Our data support the measurement of vWF mRNA in tumors to detect activated endothelium or angiogenesis. For this purpose, we developed a semi‐quantitative RT‐PCR for vWF mRNA. Preliminary results obtained with specimens from colon carcinoma and the corresponding normal colonic mucosa showed higher vWF mRNA levels in most tumors than in their normal counterparts. The differences in vWF mRNA levels were much larger than the differences in vessel counts between a tumor and the corresponding normal mucosa, indicating that high vWF mRNA levels in tumors may indeed be an early sign of activation of the endothelium. The rapidity, objectivity, sensitivity and specificity of this technique make it suitable for routine clinical application to identify aggressive, highly angiogenic tumors. Int. J. Cancer 85:281–288, 2000. ©2000 Wiley‐Liss, Inc.

Endoscopic biliary drainage before pancreaticoduodenectomy for periampullary malignancies

Despite decreased operative mortality, pancreaticoduodenectomy (PD) remains a formidable operation with substantial morbidity. We have evaluated the influence of preoperative endoscopic biliary drainage (EBD) on morbidity after PD for malignant biliary obstruction by retrospectively reviewing the medical records of 182 patients undergoing PD between April 1985 and August 1996. Of 52 study patients with malignant obstructive jaundice, 22 underwent preoperative EBD, and 30 were not drained. Eighty-three patients were excluded for bilirubin levels less than 5 mg/dl, 43 had other biliary drainage, and 4 had jaundice with benign pathology. Preoperative, intraoperative, and postoperative factors were compared. The two groups were well matched for clinical presentation and operative characteristics except for lower preoperative values of liver chemistries in patients undergoing EBD. Length of postoperative hospitalization for patients undergoing EBD was 13.5 days, compared with 19 days for patients who were not drained (p = 0.02). Patients who were not drained tended to have more overall complications (p = 0.054). Multivariate analysis revealed time to regular diet (p < 0.0001) and no preoperative drainage (p = 0.04) to be independent factors significantly increasing the length of hospitalization. Endoscopic biliary drainage before PD significantly reduced the length of postoperative hospitalization and was associated with less postoperative morbidity. Further studies, including cost analysis, are warranted.

Bone morphogenic protein-2 gene therapy for mandibular distraction osteogenesis

Abstract:Distraction osteogenesis (DO) requires a long consolidation period and has a low but real failure rate. Bone morphogenic proteins (BMPs) accelerate bone deposition in fractures and critical-sized bone defects, but their effects on mandibular DO are unknown. We investigated the effect of local delivery of adenovirus containing the gene for BMP-2 (Adbmp-2) on mandibular DO in a rat model. Rats (n = 54) were distracted to 3 mm over 6 days. At the start of consolidation (POD 10), Adbmp-2 or adenovirus containing the lacZgene (AdlacZ) was injected directly into the distraction zone. After 1, 2, and 4 weeks of consolidation, mandibles were evaluated for amount of bone deposition. Adbmp-2-treated specimens demonstrated an increased amount of new bone formation by radiographic, histologic, and histomorphometric analysis. This study demonstrates that local, adenovirally–mediated delivery of BMP-2 can increase bone deposition during DO, potentially shortening consolidation and enhancing DO in poorly healing mandibles, such as occurs postirradiation.

Topical vascular endothelial growth factor reverses delayed wound healing secondary to angiogenesis inhibitor administration.

The prevention of new blood vessel growth is an increasingly attractive strategy to limit tumor growth. However, it remains unclear whether anti‐angiogenesis approaches will impair wound healing, a process thought to be angiogenesis dependent. Results of previous studies differ as to whether angiogenesis inhibitors delay wound healing. We evaluated whether endostatin at tumor‐inhibiting doses delayed excisional wound closure. C57/BL6J mice were treated with endostatin or phosphate‐buffered solution 3 days prior to the creation of two full‐thickness wounds on the dorsum. Endostatin was administered daily until wound closure was complete. A third group received endostatin, but also had daily topical vascular endothelial growth factor applied locally to the wound. Wound area was measured daily and the wounds were analyzed for granulation tissue formation, epithelial gap, and wound vascularity. Endostatin‐treated mice showed a significant delay in wound healing. Granulation tissue formation and wound vascularity were significantly decreased, but reepithelialization was not effected. Topical vascular endothelial growth factor application to wounds in endostatin‐treated mice resulted in increased granulation tissue formation, increased wound vascularity, and wound closure approaching that of control mice. This study shows that the angiogenesis inhibitor endostatin delays wound healing and that topical vascular endothelial growth factor is effective in counteracting this effect.

Magnetic resonance cholangiopancreatography accurately predicts the presence or absence of choledocholithiasis

Accurate common bile duct (CBD) imaging in patients with biliary calculi is an important determinant of specific therapy. Noninvasive methods to evaluate calculi in the CBD have limited accuracy and rely mainly on ultrasonography and computed tomography. Magnetic resonance cholangiopancreatography (MRCP) is a new noninvasive modality available to evaluate the biliary system. This study was undertaken to assess the accuracy of MRCP in predicting the presence or absence of CBD stones in patients at increased risk for choledocholithiasis. The medical records of 48 patients with a final diagnosis of biliary calculous disease undergoing MRCP between November 1995 and April 1997 were retrospectively reviewed. Three groups were identified: choledocholithiasis (n = 19), gallstone pancreatitis (n = 11), and uncomplicated cholelithiasis (n = 18). In all patients the presence or absence of CBD calculi, as determined by MRCP, was correlated with the final diagnosis obtained from endoscopic retrograde cholangiopancreatography (ERCP) (n = 19), intraoperative cholangiography (n = 6), CBD exploration (n = 13), or clinical follow-up (n = 10). Sensitivity, specificity, and accuracy of MRCP were determined. The major clinical indications for MRCP in the 48 patients ware abnormal liver function tests followed by hyperamylasemia. Twenty patients were diagnosed with CBD stones and 28 were not. MRCP correctly predicted the presence of CBD stones in 19 of 20 patients and failed to detect CBD stones in one patient with gallstone pancreatitis. MRCP incorrectly predicted the presence of CBD stones in 3 of 28 patients ultimately found to have gallstones and no CBD stones. MRCP correctly predicted the absence of CBD stones in the other 25 patients including 10 patients with gallstone pancreatitis. Overall, MRCP had a sensitivity of 95%, a specificity of 89%, and an accuracy of 92%. MRCP is an accurate, noninvasive test for evaluating the CBD duct for the presence or absence of calculi in patients suspected of having CBD stones. Our data support the use of MRCP in the preoperative evaluation of these patients as findings may influence therapeutic decisions.

Magnetic resonance imaging with magnetic resonance cholangiopancreatography accurately predicts resectability of pancreatic carcinoma

Accurate preoperative staging of pancreatic malignancy aids in directing appropriate therapy and avoids unnecessary invasive procedures. We evaluated the accuracy of magnetic resonance imaging (MRI) with magnetic resonance cholangiopancreatography (MRCP) in determining resectability of pancreatic malignancy. Twenty-one patients with suspected pancreatic malignancy underwent dynamic, contrast-en-hanced breath-hold MRI with MRCP prior to surgical evaluation. Results of this study were correlated with operative results and pathologic findings. The sensitivity, specificity, and accuracy of MRI with MRCP in detecting a mass, determining the nature of the mass, and predicting lymph node involvement and resectability were determined. MRI with MRCP correctly identified the presence of a pancreatic mass in all 21 of these patients. Following pathologic correlation, it was determined that MRI with MRCP was 81 % accurate in determining the benign or malignant nature of the pancreatic mass and 43% accurate in predicting lymph node involvement. In predicting resectability, MRI with MRCP had a sensitivity of 100%, specificity of 83%, positive predictive value of 94%, negative predictive value of 100%, and accuracy of 95%. MRI with MRCP is an accurate, noninvasive technique in the preoperative evaluation of pancreatic malignancy. Information obtained from MRI with MRCP including identification of a mass and predicting tumor resectability may be of value in staging and avoiding unnecessary invasive diagnostic procedures in patients with pancreatic cancer.

Stem cells and distraction osteogenesis: Endothelial progenitor cells home to the ischemic generate in activation and consolidation

Background: Ischemia is a limiting factor during distraction osteogenesis. The authors sought to determine the extent of ischemia in the distraction zone and whether endothelial progenitor cells home to the distraction zone and participate in local vasculogenesis. Methods: Laser Doppler imaging was used to assess the extent of blood flow in the distraction zone in gradually distracted, immediately distracted, and osteotomized rat mandibles during activation and consolidation. Animals (n = 50; 25 rats with unilateral gradual distraction and contralateral osteotomy as an internal control, and 25 rats with unilateral immediate distraction) were examined on postoperative days 4, 6, and 8 of activation, and after 1 and 2 weeks of consolidation. Endothelial progenitor cells isolated from human peripheral blood were labeled with fluorescent DiI dye, and 0.5 × 106 cells were injected intra-arterially under direct vision into each carotid artery at the start of activation in nude rats (n = 18) that then underwent the distraction protocol outlined above. Results: Doppler flow analysis demonstrated relative ischemia during the activation period in the distraction osteogenesis group and increased blood flow in the osteotomized control group as compared with flow in a normal hemimandible [normal, 1 (standardized); distraction osteogenesis, 0.58 ± 0.05; control, 2.58 ± 0.21; p < 0.05 for both results]. We observed a significantly increased endothelial progenitor cell population at the generate site versus controls at midactivation and at 1 and 2 weeks of consolidation [25 ± 1.9 versus 1 ± 0.3 DiI-positive cells per high-power field (p < 0.05), 124 ± 21 versus 8 ± 4 DiI-positive cells per high-power field (p < 0.05), and 106 ± 18 versus 9 ± 3 DiI-positive cells per high-power field (p < 0.05), respectively]. Conclusions: These data suggest that the distraction zone becomes relatively ischemic during activation and that endothelial progenitor cells home to the ischemic generate site during the activation phase and remain during the consolidation phase. Selective expansion of these stem cells may be useful in overcoming ischemic limitations of distraction osteogenesis. Moreover, their homing capability may be used to effect site-specific transgene delivery to the generate.

Sonographically guided percutaneous carpal tunnel release: An anatomic and cadaveric study

Minimally invasive techniques have become the standard of care for multiple procedures. This paper demonstrates both the surgeons’ capacity to perform an accurate anatomic evaluation of the hand and forearm (n = 10) and the use of this anatomic information to accurately perform sonographically guided, percutaneous carpal tunnel release using a single-portal endoscope without direct or indirect visualization in a cadaver model (n = 6). Open dissection was then performed to confirm complete ligament transection and to evaluate the surrounding structures for injury. In all 6 cadavers, the transverse carpal ligament was transected completely without injury to any surrounding structures. With further investigation, this novel technique may offer a less invasive, office-based method for the surgical treatment of carpal tunnel syndrome that may offer patients an expedited recovery.

Ex vivo transduction of microvascular free flaps for localized peptide delivery.

Abstract:Gene therapy is a promising modality for the treatment of soft tissue malignancies. Our laboratory has developed a novel technique of gene transfer using microvascular free flaps that addresses many of the current barriers preventing gene therapy from achieving widespread clinical use. Our previous work has demonstrated our ability to transduce free flaps with an adenovirus encoding the reporter gene lacZ. In this current study, we show that microvascular free flaps can be transduced with an adenovirus encoding the angiogenesis inhibitor endostatin with high levels of local flap expression. These transduced free flaps were able to serve as “biologic pumps” and were able to secrete endostatin into the serum as demonstrated by enzyme-linked immunosorbent assay. This form of “biologic brachytherapy” could provide a novel approach for the continuous delivery of therapeutic genes to a localized area while avoiding many of the practical obstacles currently limiting gene therapy.

Endostatin inhibits ischemia-induced neovascularization and increases ischemic tissue loss.

The impact of inhibitors of tumor angiogenesis (endostatin, angiostatin) on the neovascularization required for the healing of transferred tissue has not been examined. We investigated the effect of endostatin on the functional neovascularization of random pattern flaps. C57BL6 mice were pretreated with endostatin beginning 3 days prior to surgery (n = 10), and daily injections continued throughout the study. Dorsal random cutaneous flaps were raised in both treatment and control (saline-treated) groups. The remaining cranial attachment was divided on day 9. Oxygen tension (PO2) was measured using a microprobe on days 1, 3, 5 and 16. Flaps were harvested and the vasculature was stained with CD31 on day 16. We found that endostatin significantly decreased flap survival. Mice that were treated with endostatin had fewer CD31+ blood vessels, worse flap perfusion at all time points, and lower oxygen tensions throughout the length of the flap. These findings have potential implications for the patients undergoing antiangiogenesis therapy who require surgical reconstruction.