Kathleen E. Rodgers

Effect of angiotensin II on hematopoietic progenitor cell proliferation.

Angiotensin II (AII) induced the proliferation of hematopoietic progenitor cells (HPC) isolated from murine bone marrow or human cord blood. The formation of colonies with more than 50 cells increased approximately five‐sevenfold in cultures of murine lineage‐negative (Lin−) bone marrow cells both in the presence (day 10) and absence (day 13) of colony‐stimulating factors (CSF). This could be blocked with addition of Losartan, an antagonist of AIITR1. The increase in proliferation of early hematopoietic progenitors (Lin−Sca l+ cells) by AII was approximately threefold and occurred only in the presence of CSF, suggesting that AII may affect mesenchymal stromal cells to induce CSF production and might directly affect early HPC. These in vitro studies were replicated with human HPC isolated from cord blood. AII also accelerated the proliferation and formation of colony‐forming units (CFU)‐granulocyte/erythroid/macrophage/megakaryocyte and CFU‐granulocyte/macrophage colonies by CD34+CD38− enriched progenitors but only in the presence of CSF. Additional studies also indicated that AII can act to increase proliferation in suspension culture. Exposure of CD34+ cells to AII in suspension culture, prior to placement in a semisolid medium with erythropoietin, increased the formation of colonies with more than 50 cells and erythroid progenitors approximately five‐ and 20‐fold, respectively. Further, mRNA for the AT1a receptor was expressed by human bone marrow CD34+CD38− cells, CD34+CD38− cells, and lymphocytes, but not mature myeloid cells. Similarly, mRNA for the AT1a receptor was expressed on human stromal cell clones, offering further support to the hypothesis that AII acts partially through the mesenchymal compartment of the bone marrow. These data suggest that AII may be a factor which stimulates the proliferation of hematopoietic progenitors.

Reduction of adhesion formation by postoperative administration of ionically cross-linked hyaluronic acid

OBJECTIVE To examine the efficacy of various formulations of hyaluronic acid (HA), including HA ionically cross-linked with trivalent iron, in animal models of adhesion formation. DESIGN Hyaluronic acid formulation of varying concentrations and cross-linked densities were prepared and evaluated in a rabbit uterine horn model and a rabbit sidewall model. SETTING ETHICON, Inc., Somerville, New Jersey. SUBJECT(S) New Zealand White rabbits. INTERVENTION(S) Test formulations were applied as intraperitoneal instillates after surgery. MAIN OUTCOME MEASURE(S) Adhesion formation was assessed at 7 and 14 days (sidewall and uterine horn model, respectively). RESULT(S) Hyaluronic acid that was not ionically cross-linked was ineffective in reducing adhesions in these models even at high viscosity, whereas the ionically cross-linked formulations of HA with trivalent iron were highly effective. Efficacy improved with increased levels of ionic cross-linking. Flowable gels, which could be delivered readily by syringe and cannula, also were effective when administered at a site remote from injury and with saline present. CONCLUSION(S) Whereas previous studies show that HA was effective in reducing adhesions peripheral to the site of injury, HA ionically cross-linked with trivalent iron was effective in reducing adhesions at all sites. From these studies, a formulation of HA ionically cross-linked with trivalent iron, 0.5% Ferric Hyaluronate Gel (LUBRICOAT; ETHICON, Inc., Somerville, NJ), was identified for subsequent clinical evaluations.

Reduction of epidural fibrosis in lumbar surgery with Oxiplex adhesion barriers of carboxymethylcellulose and polyethylene oxide.

BACKGROUND CONTEXT Postsurgical epidural adhesions and fibrosis after surgery for lumbar disc herniation are a consequence of normal wound healing. The presence of fibrosis renders reoperations risky, and in some patients fibrosis may lead to nerve root tethering. PURPOSE One approach to minimizing the risk of developing epidural adhesions is to provide a barrier between the dural membrane and the healing connective tissues. The purpose of these studies was to evaluate such a barrier device. STUDY DESIGN/SETTING In vivo investigation in an animal model at a university laboratory. PATIENT SAMPLE Rabbit. OUTCOME MEASURES Gross and histomorphic evaluation. METHODS Barriers comprised of carboxymethylcellulose (CMC) and polyethylene oxide (PEO) (Oxiplex; FzioMed, Inc., San Luis Obispo, CA) were studied as devices to reduce epidural adhesion formation in rabbit laminotomy and laminectomy models. The barriers tested were either a gel alone (gel) or a gel covered with a film (gel/film combination). Two laminotomy or laminectomy sites (depending on the surgical method) were created in each rabbit at L4 and L6. One site was treated with a CMC/PEO gel, or CMC/PEO gel/film combination, and the other site served as a surgical control. Two surgical models that differed in the extent of adhesion formation at untreated injury sites and the method of injury generation were used. RESULTS Model A, which did not incorporate dural abrasion, resulted in up to 40% adhesion-free laminectomy sites in controls. Model B, which did incorporate abrasion of the dural membrane, resulted in less than 10% adhesion-free laminotomy sites in controls. Compositions of CMC/PEO gels (2.5% to 10% PEO) and films (22.5% PEO) were tested in both models. Efficacy parameters included measuring the number of sites free of epidural fibrosis and reduction in the severity of fibrosis (adhesions). Both gels and gel/film combinations consistently reduced the frequency and the extent of epidural fibrosis in both models. Gels of CMC/PEO containing a higher content of PEO (10%) and a higher molecular weight of PEO (4.4 mD) were most effective in Model B and resulted in up to 84% laminotomy sites with minimal or no epidural fibrosis, whereas controls exhibited over 90% of the sites with epidural fibrosis. Histological evaluation of the surgical sites indicated that the reduction of epidural fibrosis was accompanied by normal bone healing. In addition, these experiments demonstrated that the gel/film combination provided no additional benefit to that obtained by the gel alone. CONCLUSIONS Gels of CMC/PEO reduced epidural fibrosis and did not impair normal heal ing.

Reduction of adhesion formation with hyaluronic acid after peritoneal surgery in rabbits

OBJECTIVE To examine the effect of hyaluronic acid, a high-molecular-weight glucosaminoglycan found in the extracellular matrix, on the formation of adhesions, a major source of postoperative complications. DESIGN The ability of hyaluronic acid to reduce adhesion formation was evaluated using a standardized rabbit model. The material was administered i.p. at the end of surgery. SETTING University laboratory. ANIMAL(S) New Zealand White female rabbits. INTERVENTION(S) Intraperitoneal administration of various formulations of hyaluronic acid at the end of surgery. MAIN OUTCOME MEASURE(S) One week after surgery, a second laparotomy was performed and the extent of adhesion formation was determined. RESULT(S) Five separate molecular weight ranges of hyaluronic acid representing eight viscosities between 1,000 and 12,000 centipoise (CPS) were shown to reduce adhesion formation in this model. All volumes, 1 to 30 mL, of hyaluronic acid tested reduced adhesion formation. In addition, the low-viscosity, low-molecular-weight hyaluronic acid significantly reduced adhesion formation when added to the trauma site or when injected at a site remote from the trauma area. CONCLUSION(S) This study showed that hyaluronic acid administered at the end of surgery reduced adhesion formation.

Effect of oxiplex* films (PEO/CMC) on adhesion formation and reformation in rabbit models and on peritoneal infection in a rat model

OBJECTIVE To assess the efficacy of Oxiplex (FzioMed, Inc., San Luis Obispo, CA) barriers. DESIGN Film of polyethylene oxide and carboxymethylcellulose (Oxiplex) were tested for strength and tissue adherence. Films were selected for evaluation in models for biocompatability and adherence. Three films were selected for evaluation in efficacy studies, and one was evaluated for effects on bacterial peritonitis. Handling characteristics of Oxiplex film were evaluated via laparoscopy. SETTING University laboratory. PATIENT(S) Rabbits, rats, pigs. INTERVENTION(S) Placement of Oxiplex prototypes at the site of injury. MAIN OUTCOME MEASURE(S) Mechanical properties, biocompatibility, tissue adherence, adhesion development, infection potentiation, and device handling. RESULT(S) Mechanical tests indicated that tensile strength and elongation were inversely correlated. All films tested had excellent tissue adherence properties. Selected films, based on residence time and biocompatibility, prevented adhesion formation in all animals and were highly efficacious in preventing adhesion reformation. The optimal Oxiplex prototype prevented adhesion reformation in 91% of the animals. This Oxiplex film, dyed to allow visualization, prevented adhesion reformation and did not affect bacterial peritonitis. In a laparoscopic model, the Oxiplex film, delivered in FilmSert forceps, via a 5.0-mm trocar, rapidly unfurled and could be easily applied to tissue with strong adherence. CONCLUSION(S) These data show development of an adhesion prevention material that is tissue adherent, can be placed via laparoscopy, and does not affect host resistance.

Development of angiotensin (1-7) as an agent to accelerate dermal repair

Angiotensin II has been shown to be a potent agent in the acceleration of wound repair. Angiotensin (1‐7), a fragment of angiotensin II that is not hypertensive, was found to be comparable to angiotensin II in accelerating dermal healing. This activity was evaluated in four models: rat and diabetic mouse full‐thickness excisional wounds; rat random flap; and guinea pig partial thickness thermal injury. In all models, angiotensin (1‐7) was comparable to angiotensin II. Angiotensin (1‐7) accelerated the closure of wounds in diabetic mice and rats. In diabetic mice the resultant tissue at day 25 after injury was more comparable to normal tissue than the fibrotic scar observed in placebo‐treated wounds. In the random flap model, angiotensin (1‐7) was comparable to angiotensin II in maintaining flap viability (approximately 82%) and flap survival (40%). Finally, angiotensin (1‐7) increased proliferation in the hair follicles at the edge of the wound and site of thermal injury, and the number of patent blood vessels on day 7 after partial thickness thermal injury. These data may be partially explained by the effect of angiotensin II and angiotensin (1‐7) on keratinocyte proliferation. While platelet‐derived growth factor had no effect on keratinocyte proliferation, angiotensin II and angiotensin (1‐7) significantly increased keratinocyte proliferation. These data show that angiotensin(1‐7) is comparable to angiotensin II in accelerating skin repair. Furthermore, the hypertensive and wound healing effects can be separated within the family of angiotensin peptides.

A rapamycin-binding protein polymer nanoparticle shows potent therapeutic activity in suppressing autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome

Sjögrens syndrome (SjS) is a chronic autoimmune disease characterized initially by lymphocytic infiltration and destruction of exocrine glands, followed by systemic organ damage and B-cell lymphoma. Conventional treatment is based on management of symptoms and there is a shortage of therapies that address the underlying causes of inflammation at source exocrine tissue. The aim of this study was to test a novel protein polymer-based platform consisting of diblock copolymers composed from Elastin-like Polypeptides (ELPs) fused with FKBP12, to deliver a potent immunosuppressant with dose-limiting toxicity, rapamycin (Rapa) also known as Sirolimus, and evaluate its effects on the inflamed lacrimal gland (LG) of non-obese diabetic mouse (NOD), a classic mouse model of SjS. Both soluble and diblock copolymer ELPs were fused to FKBP12 and characterized with respect to purity, hydrodynamic radii, drug entrapment and release. Both formulations showed successful association with Rapa; however, the nanoparticle formulation, FSI, released drug with nearly a 5 fold longer terminal half-life of 62.5h. The strong interaction of FSI nanoparticles with Rapa was confirmed in vivo by a shift in the monoexponential pharmacokinetic profile for free drug to a biexponential profile for the nanoparticle formulation. When acutely administered by injection into NOD mice via the tail vein, this FSI formulation significantly suppressed lymphocytic infiltration in the LG relative to the control group while reducing toxicity. There was also a significant effect on inflammatory and mammalian target of Rapamycin (mTOR) pathway genes in the LG and surprisingly, our nanoparticle formulation was significantly better at decreasing a proposed tear biomarker of SjS, cathepsin S (CATS) compared to free drug. These findings suggest that FSI is a promising tool for delivering Rapa for treatment of SjS in a murine model and may be further explored to meet the unmet medical challenge of SjS.

Histologic alterations in dermal repair after thermal injury effects of topical angiotensin II

Previously, we determined that quantitative assessment of epithelialization of the burn site could be performed using quantitative immunohistochemistry with an antibody to the protein cyclin. In this study, the effect of administration of angiotensin II (AII) on two histologic parameters of healing-the number of vascular channels at the burn site and the number of cells proliferating in hair follicles at the edge of the burn and within the burn-were evaluated. Beginning on day 4, vascular channels were noted within the burn site. Significantly more channels were noted in the burns treated with AII than those treated with placebo. With the exception of 3 postinjury days, this increase continued through day 17. Thereafter, the number of vascular channels peaked, and no differences were noted between control and treated burns. The number of cells proliferating in the hair follicle was also evaluated. At the edge of the burn, on average, 126 cells per microscope field (10x) were undergoing proliferation in the AII-treated burn on days 1 through 16 after burn injury. This is approximately a 50% increase over the number of cells proliferating in the placebo-treated burns. On day 12 (approximately 5 days before that observed in control burns), this AII-dependent proliferative response began to increase and peaked on day 19 at a level comparable to control. Thereafter, the proliferative response remained at this level through day 28. Within the area of the burn on days 1 through 15, 21 cells per medium power field on average (approximately 50% more than control) were undergoing proliferation. As on the edge of the burn, an AII-dependent increase in the number of cells proliferating in the hair follicles was observed during the latter phase of healing (on day 16 after the initiation of injury). However, unlike the edge of the burn, administration of AII led to a continued increase (approximately 50%) in the number of cells per field undergoing proliferation. AII increased neovascularization and cellular proliferation after burn injury. Through an increase in these two cellular events, AII may in turn accelerate healing of tissues after thermal injury.

Acceleration of dermal tissue repair by angiotensin II

Angiotensin II is a naturally occurring peptide which has been shown to possess angiogenic properties. In the studies reported here, angiotensin II was shown to increase the proliferation of cultured bovine aortic arch endothelial cells in a concentration‐dependent manner. Acute administration of angiotensin II in Hydron accelerated the repair of dermal injuries in a full‐thickness excisional rat model. Additional studies were done to determine the best vehicle for delivery of angiotensin II to a dermal injury. Several vehicles, including 10% low‐viscosity carboxymethyl cellulose, 4% medium‐viscosity carboxymethyl cellulose, and 3% high‐viscosity carboxymethyl cellulose, were found to be effective in this regard. Daily administration of angiotensin II for days 0 to 4 after injury (day 0 being the time of surgery) was determined to provide the optimal dosage for acceleration of wound repair by angiotensin II. In addition, dose‐response studies indicated that angiotensin II accelerated wound repair in a dose‐dependent fashion with 0.03 and 0.01 µg/rat/day of angiotensin II administered on days 0 to 4 being the minimally effective and no‐effect doses, respectively. Administration of 100 µg/day of angiotensin II in 10% carboxymethyl cellulose for 5 days after injury to animals with impaired healing (steroid‐ and adriamycin‐treated rats and diabetic mice) was also found to accelerate the rate of repair. In conclusion, angiotensin II accelerated the closure of full‐thickness skin injuries in a dose‐dependent manner in normal and impaired animal models.

Histological evaluation of the effects of angiotensin peptides on wound repair in diabetic mice

Abstract:  Recent studies have shown that angiotensin peptides accelerate dermal repair. Histological observation of samples taken at the termination of studies showed that the wounds treated with peptides were more mature and organized by day 25 after full thickness excision in diabetic mice. However, the mechanism by which this acceleration occurs has not been determined. In the experiments described here, the effect of angiotensin peptides [AII, A(1–7) and NorLeu3‐A(1–7)] on the quality of the healing wound was evaluated histologically. Administration of the peptides accelerated collagen deposition, re‐epithelialization and new blood vessel formation. By day 4, the percentage of the wound with collagen increased two‐ to six‐fold depending upon the peptide. The increase by angiotensin peptides continued throughout the experimental period. On days 4 and 7 (only) after injury, exposure to angiotensin peptides increased the number of blood vessels at the wound site two‐ to three‐fold. Finally, the percentage of the wound site covered with new epithelium increased after administration of angiotensin peptides. Re‐epithelialization was observed as early as day 4 in wounds treated with angiotensin peptides. The increase was greater at later time points [up to 8‐fold at day 14 with NorLeu3‐A(1–7)]. Fibroblast infiltration and proliferation occurred earlier in wounds treated with angiotensin peptides. Wounds treated with A(1–7) and NorLeu3‐A(1–7) had an increase in neutrophils and macrophages on day 4 after wounding. Overall, administration of these peptides resulted in a healing site that was more mature, including reorganization of the collagen into a basket‐weave appearance. Further, these studies confirm the superiority of NorLeu3‐A(1–7) to AII and A(1–7) in wound healing evaluated at a microscopic level.