Brian C. Cooley

a Prospective Study of Microvascular Free-flap Surgery and Outcome

&NA; Over a 6‐month period, 23 members of the International Microvascular Research Group participated in a prospective survey of their microvascular free‐flap practice. Data were recorded with each case for 60 variables covering patient characteristics, surgical technique, pharmacologic treatment, and postoperative outcome. A total of 493 free flaps were reported with a representative demographic distribution for age, sex, indications for surgery, risk factors, flap type, surgical technique, and pharmacologic intervention. Mixed effects logistic regression modeling was used to determine predictors of flap failure and associated complications. The overall incidence of flap failure was 4.1 percent (20 of 493). Reconstruction of an irradiated recipient site and the use of a skin‐grafted muscle flap were the only statistically significant predictors of flap failure, with increased odds of failure of 4.2 (p = 0.01) and 11.1 (p = 0.03), respectively. A postoperative thrombosis requiring re‐exploration surgery occurred in 9.9 percent of the flaps. The incidence of this complication was significantly higher when the flap was transferred to a chronic wound and when vein grafts were needed, with increased odds of failure of 2.9 (p = 0.02) and 2.5 (p = 0.02), respectively. There was a lower incidence of postoperative thrombosis when rectus/transverse rectus abdominis muscle (TRAM) flaps were used, where odds of failure decreased by 0.36 (p = 0.04), and when subcutaneous heparin was administered in the postoperative period, where odds decreased by 0.27 (p = 0.04). There was an overall 69‐percent salvage rate for flaps identified with a postoperative thrombosis. Intraoperative thrombosis occurred in 41 cases (8.3 percent) and was observed more frequently in myocutaneous flaps or when vein grafts were needed (5.5 and 5.0 greater odds, respectively; p < 0.001) but was not associated with higher flap failure (2 of 41 cases; 4.9‐percent failure rate). The incidence of a hematoma and/or hemorrhage was increased in obese patients and when vein grafts were needed [2.7 (p = 0.02) and 2.6 (p = 0.03) greater odds, respectively], whereas this complication was significantly decreased in muscle flaps (myocutaneous or skin‐grafted muscle), in tobacco users, when a heparinized solution was used for general wound irrigation, and when the attending surgeon performed the arterial anastomosis (in contrast to the resident or fellow on staff) (p < 0.05 for each factor). With the multivariable analysis, many factors were found not to have a significant effect on flap outcome, including the recipient site (e.g., head/neck, breast, lower limb, etc.); indications for surgery (trauma, cancer, etc.); flap transfer in extremes of age, smokers, or diabetics; arterial anastomosis with an end‐to‐end versus end‐to‐side technique; irrigation of the vessel without or with heparin added to the irrigation solution; and a wide spectrum of antithrombotic drug therapies. These results present a current baseline for free‐flap surgery to which future advances and improvements in technique and practice may be compared. (Plast. Reconstr. Surg. 102: 711, 1998.)

Brava and Autologous Fat Transfer Is a Safe and Effective Breast Augmentation Alternative: Results of a 6-year, 81-patient, Prospective Multicenter Study

Background: Breast augmentation by autologous fat transfer is an appealing alternative in need of scientific validation. Methods: In a prospective multicenter study, 81 women (age range, 17 to 63 years) wore the Brava device, a bra-like vacuum-based external tissue expander, for 4 weeks and then underwent autologous fat injection using 10 to 14 needle puncture sites into each breast in a three-dimensional fanning pattern (average, 277 ml volume injected per breast). Patients resumed Brava wear within 24 hours for 7 or more days. Pretreatment and posttreatment breast volumes were derived from three-dimensional volumetric reconstruction of magnetic resonance imaging scans, and outcomes were compared with a meta-analysis of six recent published reports on autologous fat transfer breast augmentation without expansion. Follow-up ranged from 12 months to 6 years (average, 3.7 years). Results: Breast volume was unchanged between 3 and 6 months. Seventy-one of the treated women were compliant with Brava wear and had a mean augmentation volume at 12 months of 233 ml per breast compared with 134 ml per breast in published series without Brava (p < 0.00001). Graft survival was 82 ± 18 percent compared with 55 ± 18 percent without Brava (p < 0.00001). There was a strong linear correlation (R 2 = 0.87) between pregrafting Brava expansion and the resultant breast augmentation. There were no suspicious breast masses or nodules. Magnetic resonance imaging recognized a 16 percent incidence of fat necrosis easily identified at 1-year mammographic evaluation. Conclusion: The addition of Brava expansion before autologous fat grafting leads to significantly larger breast augmentations, with more fat graft placement, higher graft survival rates, and minimal graft necrosis or complications, demonstrating high safety and efficacy for the procedure. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies

Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A. We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain-deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific alphaIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8trans mice, the bleeding phenotype of FVIIInull mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII-immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII.

TGF-β Signaling Mediates Endothelial-to-Mesenchymal Transition (EndMT) During Vein Graft Remodeling

In vivo endothelial cell fate mapping demonstrates that TGF-β signaling is a central pathway regulating the endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling. Negative Remodeling In coronary bypass surgery, veins are grafted to arteries, in hopes of generating a functional vessel. Although a routine procedure, grafting can result in a negative remodeling process—with a poorly understood underlying mechanism. Here, Cooley and colleagues linked vein graft stenosis (blood vessel narrowing) and negative remodeling to a process called the endothelial-to-mesenchymal transition (EndMT). Although well known during development, the presence of EndMT in the vasculature is less documented and therefore represents a possible new target in preventing graft failure. The authors tracked endothelial cells in mice using yellow fluorescent protein (YFP), and saw that these cells lining the vessel walls contributed to arterial thickening (neointima formation) after vein grafting by first converting to mesenchymal cells. EndMT occurred via transforming growth factor–β (TGF-β) signaling, specifically through intermediates Smad2/3 and Slug. Knowing the pathway at play is important for translation to the clinic because therapeutics can be designed to target these signaling molecules. Indeed, the authors found that blocking TGF-β with an antibody or knocking down Smad2 in vivo in mice prevented EndMT. The mesenchymal transition was also noted in failed vein grafts taken from patients, suggesting that EndMT is also present in humans and contributes to graft failure and restenosis. More testing is required in human samples to confirm the mouse data, but EndMT appears to be a viable target for improving graft outcomes after surgery in patients. Veins grafted into an arterial environment undergo a complex vascular remodeling process. Pathologic vascular remodeling often results in stenosed or occluded conduit grafts. Understanding this complex process is important for improving the outcome of patients with coronary and peripheral artery disease undergoing surgical revascularization. Using in vivo murine cell lineage–tracing models, we show that endothelial-derived cells contribute to neointimal formation through endothelial-to-mesenchymal transition (EndMT), which is dependent on early activation of the Smad2/3-Slug signaling pathway. Antagonism of transforming growth factor–β (TGF-β) signaling by TGF-β neutralizing antibody, short hairpin RNA–mediated Smad3 or Smad2 knockdown, Smad3 haploinsufficiency, or endothelial cell–specific Smad2 deletion resulted in decreased EndMT and less neointimal formation compared to controls. Histological examination of postmortem human vein graft tissue corroborated the changes observed in our mouse vein graft model, suggesting that EndMT is operative during human vein graft remodeling. These data establish that EndMT is an important mechanism underlying neointimal formation in interpositional vein grafts, and identifies the TGF-β–Smad2/3–Slug signaling pathway as a potential therapeutic target to prevent clinical vein graft stenosis.

An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

Repair of calvarial defects with flap tissue: Role of bone morphogenetic proteins and competent responding tissues

Bone morphogenetic proteins 2 through 8 have the ability to induce the in vivo transformation of extraskeletal mesenchymal tissue into bone. The aims of this investigation were to determine the optimal responding tissue and the specificity of the inductive effect of bone morphogenetic protein 3. The optimal responding tissue was found to be skeletal muscle. The specificity of this response to bone morphogenetic protein 3 was compared with that of recombinant human basic fibroblast growth factor, recombinant platelet-derived growth factor, and recombinant insulin-like growth factor. Bone morphogenetic protein 3 was the only factor that induced de novo bone formation. This ability to transform muscle into bone was tested in 7 x 7 mm irradiated skull defects in the rat. After 1500 rads of exposure, these defects showed no significant signs of healing by 8 months. When these defects were treated with the microvascular transfer of a nonirradiated muscle flap, they had 8 percent healing at 4 months and 37 percent healing by 8 months. Defects treated with 30 micrograms bone morphogenetic protein 3 (without the muscle flap) achieved 50 percent healing by 4 months and 64 percent healing by 8 months. When the defects were treated with both the muscle flap and bone morphogenetic protein 3, there was 96 percent healing by 4 months and 100 percent healing by 8 months (p < 0.015, compared with bone morphogenetic protein 3 alone at both time points). At 8 months, the transplanted muscle was entirely transformed into bone and healed the skull defect with newly generated bone indistinguishable from the surrounding calvarial tissue. These findings suggest a potential clinical utility of bone morphogenetic protein 3-induced bone formation in skeletal reconstructions. Furthermore, they also show that there is a collaborative requirement for both the osteoinductive factor bone morphogenetic protein 3 and the presence of competent responsive cells in the well-perfused muscle.

Inhibition of polyphosphate as a novel strategy for preventing thrombosis and inflammation

Inorganic polyphosphates are linear polymers of orthophosphate that modulate blood clotting and inflammation. Polyphosphate accumulates in infectious microorganisms and is secreted by activated platelets; long-chain polyphosphate in particular is an extremely potent initiator of the contact pathway, a limb of the clotting cascade important for thrombosis but dispensable for hemostasis. Polyphosphate inhibitors therefore might act as novel antithrombotic/anti-inflammatory agents with reduced bleeding side effects. Antipolyphosphate antibodies are unlikely because of polyphosphates ubiquity and simple structure; and although phosphatases such as alkaline phosphatase can digest polyphosphate, they take time and may degrade other biologically active molecules. We now identify a panel of polyphosphate inhibitors, including cationic proteins, polymers, and small molecules, and report their effectiveness in vitro and in vivo. We also compare their effectiveness against the procoagulant activity of RNA. Polyphosphate inhibitors were antithrombotic in mouse models of venous and arterial thrombosis and blocked the inflammatory effect of polyphosphate injected intradermally in mice. This study provides proof of principle for polyphosphate inhibitors as antithrombotic/anti-inflammatory agents in vitro and in vivo, with a novel mode of action compared with conventional anticoagulants.

Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A

The natural cell type(s) that synthesize and release factor VIII (FVIII) into the circulation are still not known with certainty. In vitro studies indicate that artificial expression of FVIII in endothelial cells produces an intracellular pool of FVIII that can be mobilized together with its carrier protein, von Willebrand factor (VWF), by agonists. Here, we show that expression of human B-domain deleted FVIII (hFVIII) in the vascular endothelium of otherwise FVIII-deficient mice results in costorage of FVIII and VWF in endothelial Weibel-Palade bodies and restores normal levels and activity of FVIII in plasma. Stored FVIII was mobilized into the circulation by subcutaneous administration of epinephrine. Human FVIII activity in plasma was strictly dependent on the presence of VWF. Endothelial-specific expression of hFVIII rescued the bleeding diathesis of hemophilic mice lacking endogenous FVIII. This hemostatic function of endothelial cell-derived hFVIII was suppressed in the presence of anti-FVIII inhibitory antibodies. These results suggest that targeting FVIII expression to endothelial cells may establish a releasable pool of FVIII and normalize plasma FVIII level and activity in hemophilia A, but does not prevent the inhibitory effect of anti-FVIII antibodies on the hemostatic function of transgene-derived hFVIII as is seen with platelet-derived FVIII expression.

Lentivirus‐mediated platelet gene therapy of murine hemophilia A with pre‐existing anti‐factor VIII immunity

Summary.  Background:  The development of inhibitory antibodies, referred to as inhibitors, against exogenous factor VIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof‐of‐principle that platelet‐specific expression could be successful in treating hemophilia A in the presence of inhibitory antibodies.

A phase II trial of intraluminal irrigation with recombinant human tissue factor pathway inhibitor to prevent thrombosis in free flap surgery.

A multicenter, multinational, blinded, randomized, parallel‐group, phase II study was conducted to investigate the use of recombinant human tissue factor pathway inhibitor (rhTFPI; SC‐59735) as an antithrombotic additive to the intraluminal irrigating solution during microvascular anastomosis in free flap reconstructive surgery. A total of 622 patients undergoing free flap reconstruction were randomly assigned to three groups. For each group, a different intraluminal irrigating solution was administered at completion of the microvascular arterial and venous anastomoses and before blood flow to the flap was reestablished: rhTFPI at a concentration of 0.05 or 0.15 mg/ml (low‐dose or high‐dose group, respectively) or heparin at a concentration of 100 U/ml (current‐standard‐of‐practice group). There were no other differences in treatment among the groups. Patient characteristics, risk factors, and surgical techniques used were similar among all three groups. Flap failure was lower (2 percent) in the low‐dose rhTFPI group than in the high‐dose rhT‐FPI (6 percent) and heparin (5 percent) groups, but this difference was not statistically significant (p = 0.069). There were no significant differences in the rate of intraoperative revisions of vessel anastomoses (11 percent, 12 percent, and 13 percent) or postoperative thrombosis (8 percent, 8 percent, and 7 percent) among the low‐dose rhTFPI, high‐dose rhTFPI, and heparin groups, respectively. The rate of postoperative wound hematoma was significantly lower in the low‐dose rhTFPI group (3 percent) than in the high‐dose rhTFPI (8 percent) and heparin (9 percent) groups (p = 0.040). There were no differences in blood chemistry or coagulation values among the three study groups. Other than hematomas, there were no differences in the incidence or severity of adverse reactions among the three groups. It is concluded that use of rhTFPI as an intraluminal irrigant during free flap reconstruction is safe, well tolerated, and as efficacious as use of heparin for preventing thrombotic complications during and after the operation. Furthermore, the lower dose of rhTFPI (0.05 mg/ml) may reduce the occurrence of postoperative hematoma and help prevent flap failure. (Plast. Reconstr. Surg. 107: 408, 2001.)