Arin K. Greene

Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470.

Angiogenesis is crucial for tumor growth. Angiogenesis inhibitors, such as O-(chloracetyl-carbamoyl) fumagillol (TNP-470), are thus emerging as a new class of anticancer drugs. In clinical trials, TNP-470 slowed tumor growth in patients with metastatic cancer. However, at higher doses necessary for tumor regression, many patients experienced neurotoxicity. We therefore synthesized and characterized a water-soluble conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, Gly-Phe-Leu-Gly linker and TNP-470. This conjugate accumulated selectively in tumor vessels because of the enhanced permeability and retention (EPR) effect. HPMA copolymer–TNP-470 substantially enhanced and prolonged the activity of TNP-470 in vivo in tumor and hepatectomy models. Polymer conjugation prevented TNP-470 from crossing the blood-brain barrier (BBB) and decreased its accumulation in normal organs, thereby avoiding drug-related toxicities. Treatment with TNP-470 caused weight loss and neurotoxic effects in mice, whereas treatment with the conjugate did not. This new approach for targeting angiogenesis inhibitors specifically to the tumor vasculature may provide a new strategy for the rational design of cancer therapies.

Microdeformational wound therapy: effects on angiogenesis and matrix metalloproteinases in chronic wounds of 3 debilitated patients.

The vacuum-assisted closure (VAC) device causes microdeformations of the wound surface in contact with the foam. Because angiogenesis and matrix metalloproteinase (MMP) activity are altered in chronic wounds, we hypothesized that microdeformations stimulate capillary formation and affect MMP activity. A VAC device was used to deliver microdeformational wound therapy (MDWT) to the chronic wounds of 3 debilitated patients. Debrided tissue was obtained from wound areas with and without foam contact. Microvessel density and MMP activity were determined by immunohistochemistry and zymography, respectively. Microvessel density of MDWT-treated wounds was 4.5% (±0.8) compared with areas not covered by foam [1.6% (±0.1)] (P = 0.05) during the first week of treatment and 2.7% (±0.3) compared with untreated tissue [1.3% (±0.1)] (P = 0.03) during the second treatment week. Wounds subjected to MDWT had greater microvessel density compared with the same wound prior to treatment [1.5% (±0.3)] (P = 0.02). MMP-9/NGAL (neutrophil gelatinase-associated lipocalin), MMP-9, latent MMP-2, and active MMP-2 were reduced by 15%–76% in MDWT-treated wounds. MDWT provides a favorable wound-healing environment by increasing angiogenesis and decreasing MMP activity in chronic wounds.

Partial hepatectomy in the mouse: technique and perioperative management.

Hepatic surgery in mice is challenging because of the delicate nature of the liver, lack of intravenous access, and risk of hemorrhage. In order to study the ability of the liver to regenerate after surgical resection, we developed a novel, rapid, and safe technique for partial hepatectomy in mice. We determined the relative contributions of the seven lobes of the mouse liver and resected the three most anterior lobes for a 68% hepatectomy. We used general anesthesia, a small upper midline incision, silk suture to tie off the lobes to be resected, warming pads and lights, as well as subcutaneous saline injection to ensure minimal morbidity. We have performed a safe two-thirds hepatic resection in 288 of 300 C57BL6 mice (96%). Perioperative mortality was due to technical error. Minimal long-term morbidity was appreciated. This technique may be applied to any type of hepatic resection in mice. In addition, the general operative technique and perioperative management of these mice may be applied to all types of murine intra-abdominal procedures used for surgical research.

Lymphatic and Other Vascular Malformative/Overgrowth Disorders Are Caused by Somatic Mutations in PIK3CA

OBJECTIVES To test the hypothesis that somatic phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha (PIK3CA) mutations would be found in patients with more common disorders including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS). STUDY DESIGN We used next generation sequencing, droplet digital polymerase chain reaction, and single molecule molecular inversion probes to search for somatic PIK3CA mutations in affected tissue from patients seen at Boston Childrens Hospital who had an isolated LM (n = 17), KTS (n = 21), fibro-adipose vascular anomaly (n = 8), or congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (n = 33), the disorder for which we first identified somatic PIK3CA mutations. We also screened 5 of the more common PIK3CA mutations in a second cohort of patients with LM (n = 31) from Seattle Childrens Hospital. RESULTS Most individuals from Boston Childrens Hospital who had isolated LM (16/17) or LM as part of a syndrome, such as KTS (19/21), fibro-adipose vascular anomaly (5/8), and congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (31/33) were somatic mosaic for PIK3CA mutations, with 5 specific PIK3CA mutations accounting for ∼ 80% of cases. Seventy-four percent of patients with LM from Seattle Childrens Hospital also were somatic mosaic for 1 of 5 specific PIK3CA mutations. Many affected tissue specimens from both cohorts contained fewer than 10% mutant cells. CONCLUSIONS Somatic PIK3CA mutations are the most common cause of isolated LMs and disorders in which LM is a component feature. Five PIK3CA mutations account for most cases. The search for causal mutations requires sampling of affected tissues and techniques that are capable of detecting low-level somatic mosaicism because the abundance of mutant cells in a malformed tissue can be low.

Extracranial Arteriovenous Malformations: Natural Progression and Recurrence after Treatment

BACKGROUND Arteriovenous malformation is a dynamic vascular anomaly; it expands with age and after treatment. This study analyzed the pattern of arteriovenous malformation progression and frequency of recurrence after therapy. METHODS Patients with cutaneous and soft-tissue arteriovenous malformation were reviewed. Progression was defined as advancement to a higher Schobinger stage (I through IV) before treatment. Recurrence was defined as expansion following embolization or resection. The effect of sex, location, size, adolescence, pregnancy, and stage on progression or recurrence was analyzed. RESULTS The study included 272 patients. Children with stage I arteriovenous malformation had a 43.8 percent risk of progression before adolescence and an 82.6 percent risk before adulthood; the remaining children had progression in adulthood. Progression was more common during adolescence (56.0 percent; 95 percent confidence interval, 46.5 to 65.2) compared with childhood (38.8 percent; 95 percent confidence interval, 32.4 to 45.4) (p = 0.002). The average age at progression was 12.7 +/- 11.1 years. Diffuse arteriovenous malformations were more likely to progress compared with localized lesions (p < 0.001). Sex (p = 0.46), location (p = 0.36), and pregnancy (p = 0.20) did not influence expansion. Resection (with or without embolization) had a lower recurrence rate (81 percent) and longer time to recurrence (42.7 percent >1 year), compared with embolization alone (98 percent and 14.4 percent >1 year, respectively) (p < 0.001). Recurrence was less likely when lower staged lesions were treated (p < 0.001) and did not correlate with sex (p = 0.10), location (p = 0.60), size (p = 0.07), or age (p = 0.21). CONCLUSIONS Arteriovenous malformation is likely to progress before adulthood, particularly during adolescence, and usually reexpands following treatment. Resection (with or without embolization) for lower staged or localized arteriovenous malformation offers the best chance for long-term control.

Endothelial-Directed Hepatic Regeneration after Partial Hepatectomy

ObjectiveTo determine the role of the microvascular endothelium in the regulation of regenerating liver mass after partial hepatectomy. Summary Background DataAngiogenesis is critical for both pathologic and physiologic processes. The ability of certain tissues, such as the liver, kidney, and spleen, to regenerate after injury is poorly understood. The liver will regenerate to its normal mass within 8 days of surgical excision. Because the authors have previously shown that the endothelial cell regulates tumor mass, we hypothesized that normal adult organ mass is also controlled by the endothelial cell. MethodsTwo-thirds partial hepatectomy was performed in 7- to 8-week-old C57 BL/6 mice, followed by systemic treatment with either the angiogenesis stimulator basic fibroblast growth factor (bFGF) (1 &mgr;g/g/d intraperitoneal) or the angiogenesis inhibitor TNP-470 (30 mg/kg/qod subcutaneous). Groups of three mice were then euthanized on postoperative days 2, 4, 6, and 8, and the livers were weighed and analyzed by immunohistochemistry. ResultsbFGF accelerated hepatic regeneration by 42%, 19%, 16%, and 16% on postoperative days 2, 4, 6, and 8, respectively. TNP-470 inhibited hepatic regeneration by 46%, 74%, 67%, and 64% on postoperative days 2, 4, 6, and 8, respectively. Immunohistochemistry revealed that bFGF and TNP-470 primarily affected the endothelial compartment. Specifically, bFGF increased endothelial proliferation and decreased endothelial apoptosis. TNP-470, in contrast, inhibited endothelial cell proliferation. The cessation of the regenerative process correlated with a decrease in endothelial proliferation and an increase in endothelial apoptosis. ConclusionsThe systemic administration of angiogenesis agents modulates the regeneration of hepatic mass primarily by affecting endothelial cell proliferation or apoptosis. Endothelial cell apoptosis is associated with the cessation of the regenerative process in control mice. These results suggest that the endothelial cell is one of the key mediators of regenerating adult tissue mass in this partial hepatectomy model.

Autogenous bone graft: basic science and clinical implications.

No single biomaterial is optimum for every craniomaxillofacial application. Instead, surgeons should consider the advantages and disadvantages of each alternative in a given clinical situation, and select the material with lowest overall cost and morbidity, and the highest likelihood of success. Autogenous bone is still considered the gold standard for most applications; it becomes vascularized and osseointegrates with surrounding bone, thus minimizing the risk of infection, dislodgement, or break-down. Limitations include added operative time for graft harvest, donor site morbidity, graft resorption, molding challenges, and limited availability, especially in the pediatric population. Numerous alternatives to bone graft have become available to address these limitations; unfortunately, most of these products are expensive, do not osseointegrate, and have unpredictable biologic activity. Understanding the physiologic behavior of autogenous bone graft can help clarify the indications for its use and provide a conceptual framework for achieving the best possible outcome when this alternative is chosen.

Prevention of intra-abdominal adhesions using the antiangiogenic COX-2 inhibitor celecoxib.

Objective:To determine the effects of COX-2 specific inhibitors on postoperative adhesion formation. Summary and Background Data:Intra-abdominal adhesions are the major cause of intestinal obstruction and secondary infertility after surgical procedures. Because adhesion synthesis requires angiogenesis, and cyclooxygenase-2 enzyme (COX-2) inhibitors have antiendothelial activity, we tested COX-2 inhibitors in a murine model of intra-abdominal adhesion formation. Methods:A silicone patch was secured to the lateral abdominal wall of groups of C57BL/6 mice, followed by cecal abrasion to promote adhesion formation. Beginning on the day of surgery, mice were treated with the selective COX-2 agents, celecoxib or rofecoxib, and the nonspecific COX inhibitors, aspirin, naproxen, ibuprofen, or indomethacin. Animals were treated for 10 days and killed. A second group (celecoxib, rofecoxib, aspirin) was treated for 10 days and observed for an additional 25 days. After treatment, intra-abdominal adhesions were scored using a standard method. The patch was subjected to immunohistochemistry with the endothelial-specific marker, CD31. Results:Animals treated with selective and nonselective COX-2 inhibitors, except aspirin, had significantly fewer adhesions than control animals. Celecoxib produced a maximal reduction in adhesion formation compared with rofecoxib and the nonselective COX-2 inhibitors at 10 days. After 25 days, celecoxib and rofecoxib, but not aspirin, had fewer adhesions than control mice. Adhesions from mice treated with celecoxib had reduced microvessel density compared with rofecoxib, the nonselective COX inhibitors, and control animals. Conclusions:Selective COX-2 inhibitors, in particular celecoxib, provide durable inhibition of intra-abdominal adhesions through an antiangiogenic mechanism.

Lower-Extremity Lymphedema and Elevated Body-Mass Index

This letter reports lymphedema in 5 of 15 morbidly obese patients and suggests that morbid obesity may constitute a novel cause of lymphedema.

Management of parotid hemangioma in 100 children.

Most problematic infantile hemangiomas are successfully treated with pharmacological therapy. However, there are reports that hemangioma of the parotid gland responds poorly to corticosteroid and interferon. To better clarify the management of parotid hemangioma, the authors retrospectively studied the records of 100 consecutive patients, seen between 1975 and 2002. The characteristics of the tumor, including sex ratio, presence at birth, size, side, complications, and involvement of adjacent structures, were recorded. The indications for and response to treatment and the need for surgical procedures were documented and statistically analyzed. The female-to-male ratio was 4.5:1. Forty percent of parotid hemangiomas were on the right side, 36 percent were on the left, and 24 percent were bilateral. Forty-five percent of patients had a premonitory cutaneous lesion at birth. Fifty-nine percent of parotid hemangiomas ulcerated during the early proliferative phase. Eighty-eight percent involved nearby structures (ear, 70 percent; lip, 34 percent; subglottic region, 21 percent; eye, 18 percent; and nose, 3 percent). Seven percent of patients required tracheostomy, and 3 percent had signs of congestive heart failure. Seventy infants received pharmacological treatment. Sixty-seven patients were initially managed with corticosteroids; regression or stabilization was noted in 83 percent of tumors (56 of 67 tumors). Twenty-one patients received interferon: 11 in whom corticosteroid therapy had failed, seven in whom the tumor stabilized with corticosteroid therapy but further regression was needed, and three who had interferon as primary therapy. Ninety-five percent of the lesions that were resistant to corticosteroid subsequently responded to interferon alfa-2a or −2b. The overall response rate to pharmacological therapy was 98 percent. A reconstructive procedure was necessary during the involuting or involuted phase in 66 percent of patients: 92 percent had preauricular excision of redundant skin and/or fibrofatty tissue and 37 percent of patients had auricular revision. In summary, drug therapy was effective in the majority of infants with parotid hemangioma, whether given because the tumor was large, deforming, ulcerated, or involved nearby structures with functional consequences. Infantile hemangioma in the parotid gland responded to pharmacological treatment in a similar manner as hemangioma in other locations.