Biochemical and behavioural profile of NTBC treated Tyrosinemie type 1 mice.

BACKGROUND: Tyrosinemia type 1 (HT1) is a rare metabolic disorder caused by a defect in the tyrosine catabolic pathway. Since HT1 patients are treated with NTBC, outcome improved and life expectancy greatly increased. However extensive neurocognitive and behavioural problems have been described, which might be related to treatment with NTBC, the biochemical changes induced by NTBC, or metabolites accumulating due to the enzymatic defect characterizing the disease. OBJECTIVE: To study the possible pathophysiological mechanisms of brain dysfunction in HT1, we assessed blood and brain LNAA, and brain monoamine neurotransmitter metabolite levels in relation to behavioural and cognitive performance of HT1 mice. DESIGN: C57BL/6 littermates were divided in three different experimental groups: HT1, heterozygous and wild-type mice (n = 10; 5 male). All groups were treated with NTBC and underwent cognitive and behavioural testing. One week after behavioural testing, blood and brain material were collected to measure amino acid profiles and brain monoaminergic neurotransmitter levels. RESULTS: Irrespective of the genetic background, NTBC treatment resulted in a clear increase in brain tyrosine levels, whereas all other brain LNAA levels tended to be lower than their reference values. Despite these changes in blood and brain biochemistry, no significant differences in brain monoamine neurotransmitter (metabolites) were found and all mice showed normal behaviour and learning and memory. CONCLUSION: Despite the biochemical changes, NTBC and genotype of the mice were not associated with poorer behavioural and cognitive function of the mice. Further research involving dietary treatment of FAH-/- are warranted to investigate whether this reveals the cognitive impairments that have been seen in treated HT1 patients.

Modeling the cognitive effects of diet discontinuation in adults with phenylketonuria (PKU) using pegvaliase therapy in PAH-deficient mice.

Existing phenylalanine hydroxylase (PAH)-deficient mice strains are useful models of untreated or late-treated human phenylketonuria (PKU), as most contemporary therapies can only be initiated after weaning and the pups have already suffered irreversible consequences of chronic hyperphenylalaninemia (HPA) during early brain development. Therefore, we sought to evaluate whether enzyme substitution therapy with pegvaliase initiated near birth and administered repetitively to C57Bl/6-Pahenu2/enu2 mice would prevent HPA-related behavioral and cognitive deficits and form a model for early-treated PKU. The main results of three reported experiments are: 1) lifelong weekly pegvaliase treatment prevented the cognitive deficits associated with HPA in contrast to persisting deficits in mice treated with pegvaliase only as adults. 2) Cognitive deficits reappear in mice treated with weekly pegvaliase from birth but in which pegvaliase is discontinued at 3 months age. 3) Twice weekly pegvaliase injection also prevented cognitive deficits but again cognitive deficits emerged in early-treated animals following discontinuation of pegvaliase treatment during adulthood, particularly in females. In all studies, pegvaliase treatment was associated with complete correction of brain monoamine neurotransmitter content and with improved overall growth of the mice as measured by body weight. Mean total brain weight however remained low in all PAH deficient mice regardless of treatment. Application of enzyme substitution therapy with pegvaliase, initiated near birth and continued into adulthood, to PAH-deficient Pahenu2/enu2 mice models contemporary early-treated human PKU. This model will be useful for exploring the differential pathophysiologic effects of HPA at different developmental stages of the murine brain.

Therapeutic liver repopulation by transient acetaminophen selection of gene-modified hepatocytes.

Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we developed a method to expand hepatocytes bearing therapeutic transgenes. The common fever medicine acetaminophen becomes hepatotoxic via cytochrome p450 metabolism. Lentiviral vectors with transgenes linked in cis to a Cypor shRNA were administered to neonatal mice. Hepatocytes lacking the essential cofactor of Cyp enzymes, NADPH-cytochrome p450 reductase (Cypor), were selected in vivo by acetaminophen administration, replacing up to 50% of the hepatic mass. Acetaminophen treatment of the mice resulted in over 30-fold expansion of transgene-bearing hepatocytes and achieved therapeutic thresholds in hemophilia B and phenylketonuria. We conclude that therapeutically modified hepatocytes can be selected safely and efficiently in preclinical models with a transient regimen of moderately hepatotoxic acetaminophen.

Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing.

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele.

A novel Pah-exon1 deleted murine model of phenylalanine hydroxylase (PAH) deficiency.

Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.

AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria.

Phenylketonuria (PKU) due to recessively inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia, which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake. In this study, liver-tropic recombinant AAV2/8 vectors were used to deliver CRISPR/Cas9 machinery and facilitate correction of the Pah enu2 allele by homologous recombination. Additionally, a non-homologous end joining (NHEJ) inhibitor, vanillin, was co-administered with the viral drug to promote homology-directed repair (HDR) with the AAV-provided repair template. This combinatorial drug administration allowed for lifelong, permanent correction of the Pah enu2 allele in a portion of treated hepatocytes of mice with PKU, yielding partial restoration of liver PAH activity, substantial reduction of blood phenylalanine, and prevention of maternal PKU effects during breeding. This work reveals that CRISPR/Cas9 gene editing is a promising tool for permanent PKU gene editing.

Cardiac tissue citric acid cycle intermediates in exercised very long-chain acyl-CoA dehydrogenase-deficient mice fed triheptanoin or medium-chain triglyceride.

Anaplerotic odd-chain fatty acid supplementation has been suggested as an approach to replenish citric acid cycle intermediate (CACi) pools and facilitate adenosine triphosphate (ATP) production in subjects with long-chain fatty acid oxidation disorders, but the evidence that cellular CACi depletion exists and that repletion occurs following anaplerotic substrate supplementation is limited. We exercised very long-chain acyl-CoA dehydrogenase-deficient (VLCAD-/-) and wild-type (WT) mice to exhaustion and collected cardiac tissue for measurement of CACi by targeted metabolomics. In a second experimental group, VLCAD-/- and WT mice that had been fed chow prepared with either medium-chain triglyceride (MCT) oil or triheptanoin for 4 weeks were exercised for 60 minutes. VLCAD-/- mice exhibited lower succinate in cardiac muscle at exhaustion than WT mice suggesting lower CACi in VLCAD-/- with prolonged exercise. In mice fed either MCT or triheptanoin, succinate and malate were greater in VLCAD-/- mice fed triheptanoin compared to VLCAD-/- animals fed MCT but lower than WT mice fed triheptanoin. Long-chain odd acylcarnitines such as C19 were elevated in VLCAD-/- and WT mice fed triheptanoin suggesting some elongation of the heptanoate, but it is unknown what proportion of heptanoate was oxidized vs elongated. Prolonged exercise was associated with decreased cardiac muscle succinate in VLCAD-/- mice in comparison to WT mice. VLCAD-/- fed triheptanoin had increased succinate compared to VLCAD-/- mice fed MCT but lower than WT mice fed triheptanoin. Cardiac CACi were higher following dietary ingestion of an anaplerotic substrate, triheptanoin, in comparison to MCT.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH4) treatment in Pahenu1/enu2 (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type. We found that brain L-tyrosine, L-tryptophan, BH4 cofactor and catecholamine concentrations, and brain tyrosine hydroxylase (TH) activity were normal in these mice but that brain serotonin, 5-hydroxyindolacetic acid (5HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) content, and brain TH protein, as well as tryptophan hydroxylase type 2 (TPH2) protein levels and activity were reduced in comparison to wild-type mice. Parenteral L-Phe loading conditions did not lead to significant changes in brain neurometabolite concentrations. Remarkably, enteral BH4 treatment, which normalized brain L-Phe levels in ENU1/2 mice, lead to only partial recovery of brain serotonin and 5HIAA concentrations. Furthermore, indirect evidence indicated that the GTP cyclohydrolase I (GTPCH) feedback regulatory protein (GFRP) complex may be a sensor for brain L-Phe elevation to ameliorate the toxic effects of HPA. We conclude that BH4 treatment of HPA toward systemic L-Phe lowering reverses elevated brain L-Phe content but the recovery of TPH2 protein and activity as well as serotonin levels is suboptimal, indicating that patients with mild HPA and mood problems (depression or anxiety) treated with the current diet may benefit from supplementation with BH4 and 5-OH-tryptophan.

Blood phenylalanine reduction corrects CNS dopamine and serotonin deficiencies and partially improves behavioral performance in adult phenylketonuric mice.

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters dopamine and serotonin have been implicated in the pathophysiology of neuropsychiatric dysfunction in human phenylketonuria (PKU). In this study, we confirmed the occurrence of brain dopamine and serotonin deficiencies in association with severe behavioral alterations and cognitive impairments in hyperphenylalaninemic C57BL/6-Pahenu2/enu2 mice, a model of human PKU. Phenylalanine-reducing treatments, including either dietary phenylalanine restriction or liver-directed gene therapy, initiated during adulthood were associated with increased brain monoamine content along with improvements in nesting behavior but without a change in the severe cognitive deficits exhibited by these mice. At euthanasia, there was in Pahenu2/enu2 brain a significant reduction in the protein abundance and maximally stimulated activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2), the rate limiting enzymes catalyzing neuronal dopamine and serotonin synthesis respectively, in comparison to levels seen in wild type brain. Phenylalanine-reducing treatments initiated during adulthood did not affect brain TH or TPH2 content or maximal activity. Despite this apparent fixed deficit in striatal TH and TPH2 activities, initiation of phenylalanine-reducing treatments yielded substantial correction of brain monoamine neurotransmitter content, suggesting that phenylalanine-mediated competitive inhibition of already constitutively reduced TH and TPH2 activities is the primary cause of brain monoamine deficiency in Pahenu2 mouse brain. We propose that CNS monoamine deficiency may be the cause of the partially reversible adverse behavioral effects associated with chronic HPA in Pahenu2 mice, but that phenylalanine-reducing treatments initiated during adulthood are unable to correct the neuropathology and attendant cognitive deficits that develop during juvenile life in late-treated Pahenu2/enu2 mice.

High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU).

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters, dopamine and serotonin, have been implicated in the pathophysiology of neuropsychiatric dysfunction in phenylketonuria (PKU). Increased brain phenylalanine concentration likely competitively inhibits the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), the rate limiting steps in dopamine and serotonin synthesis respectively. Tetrahydrobiopterin (BH4) is a required cofactor for TH and TPH activity. Our hypothesis was that treatment of hyperphenylalaninemic Pah(enu2/enu2) mice, a model of human PKU, with sapropterin dihydrochloride, a synthetic form of BH4, would stimulate TH and TPH activities leading to improved dopamine and serotonin synthesis despite persistently elevated brain phenylalanine. Sapropterin (20, 40, or 100mg/kg body weight in 1% ascorbic acid) was administered daily for 4 days by oral gavage to Pah(enu2/enu2) mice followed by measurement of brain biopterin, phenylalanine, tyrosine, tryptophan and monoamine neurotransmitter content. A significant increase in brain biopterin content was detected only in mice that had received the highest sapropterin dose, 100mg/kg. Blood and brain phenylalanine concentrations were unchanged by sapropterin therapy. Sapropterin therapy also did not alter the absolute amounts of dopamine and serotonin in brain but was associated with increased homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), dopamine and serotonin metabolites respectively, in both wild type and Pah(enu2/enu2) mice. Oral sapropterin therapy likely does not directly affect central nervous system monoamine synthesis in either wild type or hyperphenylalaninemic mice but may stimulate synaptic neurotransmitter release and subsequent metabolism.

Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU).

Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU.

The Influence of Vitamin D Metabolism on Gene Expression, Matrix Production and Mineralization During Osteoprecursor Cell-Based Bone Development.

BACKGROUND: Multipotential precursor cell lines derived from human bone marrow, capable of differentiating into cartilage or bone, may provide a useful tissue development model for studying the regulation and metabolism of putative growth and differentiation factors necessary for tissue regeneration. In mammals, the process of bone development depends on the proliferation and differentiation of osteoblast lineage cells, and the subsequent synthesis and mineralization of bone extracellular matrix (ECM). Vitamin D metabolites play a pivotal role in bone and mineral homeostasis, and are positive factors on bone development. Recently, it was demonstrated that a human-derived engineered osteoblast precursor cell line (OPC1), derived from human bone marrow, can metabolize the parental precursor vitamin D3 (vitaD3) to the active steroid 1α,25-dihydroxyvitamin D3 (1,25OH2D3), and elicit an osteogenic response that results in the decrease in proliferation and increase in ECM synthesis during early bone development. The aim in this study is to characterize gene expression, matrix production and mineralization within a bone development model. METHODS: We investigated whether vitaD3 influences bone ECM mineralization in the same manner as 1,25OH2D3 in confluent cultures of OPC1s. In addition, we explored the influence of vitamin D metabolites, in combination with other commonly used osteogenic factors, ascorbic acid, ß-glycerophosphate, dexamethasone (dex) and recombinant human bone morphogenetic protein-2 (rhBMP-2) on the osteoinduction of OPC1. RESULTS: It was demonstrated that OPC1 expresses the mRNA for the enzymatic equipment necessary to convert vitaD3 to 1,25OH2D3, as well as the mRNA expression of the catabolic enzyme known to regulate the concentration of active 1,25OH2D3. It was also demonstrated that mRNA expression for the vitamin D receptor (VDR) was influenced by both vitaD3 and 1,25OH2D3. Differential results using vitamin D metabolites in combination with ascorbic acid, ß-glycerophosphate, dex and/or rhBMP-2 were observed in alkaline phosphatase (ALP) activity and calcium deposition, and mRNA expression of procollagen type I (proColI), osteocalcin (OC) and osteopontin (OP). CONCLUSIONS: Overall it was demonstrated that vitamin D in combination with osteogenic factors influences the temporal bone development sequence in a positive manner.

Extrahepatic 25-Hydroxylation of Vitamin D3 in an Engineered Osteoblast Precursor Cell Line Exploring the Influence on Cellular Proliferation and Matrix Maturation during Bone Development.

Osteoblastic precursors experience distinct stages during differentiation and bone development, which include proliferation, extracellular matrix (ECM) maturation, and ECM mineralization. It is well known that vitamin D plays a large role in the regulation of bone mineralization and homeostasis via the endocrine system. The activation of vitamin D requires two sequential hydroxylation steps, first in the kidney and then in the liver, in order to carry out its role in calcium homeostasis. Recent research has demonstrated that human-derived mesenchymal stem cells (MSCs) and osteoblasts can metabolize the immediate vitamin D precursor 25-dihydroxyvitamin D3 (25OHD3) to the active steroid lα,25-dihydroxyvitamin D3 (1,25OH2D3) and elicit an osteogenic response. However, reports of extrahepatic metabolism of vitamin D3, the parental vitamin D precursor, have been limited. In this study, we investigated whether osteoblast precursors have the capacity to convert vitamin D3 to 1,25OH2D3 and examined the potential of vitamin D3 to induce 1,25OH2D3 associated biological activities in osteoblast precursors. It was demonstrated that the engineered osteoblast precursor derived from human marrow (OPC1) is capable of metabolizing vitamin D3 to 1,25OH2D3 in a dose-dependent manner. It was also demonstrated that administration of vitamin D3 leads to the increase in alkaline phosphatase (ALP) activity associated with osteoblast ECM maturation and calcium deposits and a decrease in cellular proliferation in both osteoblast precursor cell lines 0PC1 andOMC3T3-E1. These findings provide a two-dimensional culture foundation for future three-dimensional engineered tissue studies using the OPC1 cell line.

Hepatocytes from wild-type or heterozygous donors are equally effective in achieving successful therapeutic liver repopulation in murine phenylketonuria (PKU).

Successful restoration of phenylalanine (Phe) clearance following liver-directed gene therapy in murine phenylketonuria (PKU) is likely dependent upon both the number of cells successfully transduced and the amount of phenylalanine hydroxylase (PAH) activity expressed per cell. At low levels of transduction, Phe clearance could be limited by the low absolute number of PAH-expressing cells rather than the total amount of PAH activity produced in the liver. We have evaluated the interrelationship between the number of PAH positive cells, the amount of PAH activity produced and Phe clearance through experiments with hepatocyte-mediated therapeutic liver repopulation in the Pah(enu2) mouse, a model of PKU. We compared the therapeutic efficacy of transplantation with either wild-type hepatocytes or hepatocytes from heterozygous Pah(enu2/+) donors into PAH deficient, hyperphenylalaninemic Pah(enu2)/Pah(enu2) mice. The recipient mice were also homozygous for fumarylacetoacetate hydrolase (FAH) deficiency. In this model system, FAH positive donor hepatocytes enjoy a selective growth advantage in the FAH-deficient recipient. If Phe clearance is governed predominantly by the total PAH activity, then more heterozygous cells, which express lower PAH activity than wild-type cells, should be required to correct Phe clearance. If the absolute donor cell number is more important, then wild-type hepatocytes should have no advantage over heterozygous cells. We successfully carried out therapeutic liver repopulation with heterozygous donor cells in fifteen mice and an additional thirteen transplants with wild-type cells. Blood Phe was successfully reduced in both transplant groups, and the relationship between the final blood Phe level and the extent of liver repopulation with donor cells did not differ between the two donor groups. Regardless of the type of donor cell, liver repopulation of approximately 3-10% was sufficient to at least partially reduce blood phenylalanine, and blood Phe levels were completely corrected in mice that had attained greater than approximately 10% liver repopulation. We conclude from our study that the absolute number of PAH-expressing cells likely governs Phe clearance at least at the levels of repopulation reported here and that the amount of PAH activity per donor cell is a less critical variable. The implication for liver-directed gene therapy of PKU is that only partial correction of cellular PAH deficiency may yet improve Phe clearance as long as a sufficient number of hepatocytes is successfully transduced.

Three-Dimensional Culture of Cells and Matrix Biomolecules for Engineered Tissue Development and Biokinetics Model Validation.

There has been considerable progress in cellular and molecular engineering due to recent advances in multiscale technology. Such technologies allow controlled manipulation of physiochemical interactions among cells in tissue culture. In particular, a novel chemomechanical bioreactor has recently been designed for the study of bone and cartilage tissue development, with particular focus on extracellular matrix formation. The bioreactor is equally significant as a tool for validation of mathematical models that explore biokinetic regulatory thresholds (Saha, A. K., and Kohles, S. S., 2010, "A Distinct Catabolic to Anabolic Threshold Due to Single-Cell Nanomechanical Stimulation in a Cartilage Biokinetics Model," J. Nanotechnol. Eng. Med., 1(3), p. 031005; 2010, "Periodic Nanomechanical Stimulation in a Biokinetics Model Identifying Anabolic and Catabolic Pathways Associated With Cartilage Matrix Homeostasis," J. Nanotechnol. Eng. Med., 1(4), p. 041001). In the current study, three-dimensional culture protocols are described for maintaining the cellular and biomolecular constituents within defined parameters. Preliminary validation of the bioreactor's form and function, expected bioassays of the resulting matrix components, and application to biokinetic models are described. This approach provides a framework for future detailed explorations combining multiscale experimental and mathematical analyses, at nanoscale sensitivity, to describe cell and biomolecule dynamics in different environmental regimes.

Evaluating revascularization and flap survival using vascular endothelial growth factor in an irradiated rat model.

OBJECTIVE: To evaluate the role of vascular endothelial growth factor (VEGF) plasmid DNA (pDNA) in improving flap revascularization in a previously developed rat model. Our hypothesis was that the uptake and expression of VEGF pDNA in the wound bed would improve revascularization and flap viability. DESIGN: Twenty-eight male Sprague-Dawley rats received a total dose of 40 Gy electron beam radiation to the ventral abdominal wall. After a recovery period, they underwent a ventral fasciocutaneous flap procedure with a 2-hour ischemia period. Group 1 (n = 14) received topical VEGF pDNA, in vivo cationic polymer, and fibrin sealant. Group 2 (n = 14) received topical cationic polymer and fibrin sealant only. Seven of the rats from each group underwent pedicle ligation at 8 or 14 days. The primary outcome measure was percentage of flap revascularization 5 days after pedicle ligation. RESULTS: Rats receiving VEGF pDNA had a significantly higher rate of flap revascularization (90.8% vs 79.8%) after pedicle ligation at 14 days (P = .045). At 8 days, rats receiving VEGF pDNA (group 1) had an increased rate of flap revascularization (58.2% vs 42.8%) that approached significance (P = .11). CONCLUSION: This study demonstrates the potential of VEGF pDNA to improve revascularization and flap viability in previously irradiated tissue.

Manipulation of Suspended Single Cells by Microfluidics and Optical Tweezers.

Chondrocytes and osteoblasts experience multiple stresses in vivo. The optimum mechanical conditions for cell health are not fully understood. This paper describes the optical and microfluidic mechanical manipulation of single suspended cells enabled by the µPIVOT, an integrated micron resolution particle image velocimeter (µPIV) and dual optical tweezers instrument (OT). In this study, we examine the viability and trap stiffness of cartilage cells, identify the maximum fluid-induced stresses possible in uniform and extensional flows, and compare the deformation characteristics of bone and muscle cells. These results indicate cell photodamage of chondrocytes is negligible for at least 20 min for laser powers below 30 mW, a dead cell presents less resistance to internal organelle rearrangement and deforms globally more than a viable cell, the maximum fluid-induced shear stresses are limited to ~15 mPa for uniform flows but may exceed 1 Pa for extensional flows, and osteoblasts show no deformation for shear stresses up to 250 mPa while myoblasts are more easily deformed and exhibit a modulated response to increasing stress. This suggests that global and/or local stresses can be applied to single cells without physical contact. Coupled with microfluidic sensors, these manipulations may provide unique methods to explore single cell biomechanics.

Development of an irradiated rodent model to study flap revascularization.

OBJECTIVE: To develop a reproducible free-flap animal model to study the effects of irradiation on flap revascularization. DESIGN: After institutional animal care and use committee review and approval, 16 Sprague-Dawley rats were subjected to either 23- or 40-Gy electron beam irradiation to their ventral abdominal wall. After a recovery period, the animals then underwent a ventral fasciocutaneous flap pedicled on the inferior epigastric vessels with subsequent pedicle ligation at 10 days. An additional 16 rats were subjected to 40 Gy of irradiation and underwent pedicle ligation at 8 or 14 days postoperatively to determine if time to pedicle ligation affected percentage of flap viability. RESULTS: Rats receiving 23 Gy of irradiation had the same viability as rats undergoing no radiation. Rats receiving 40 Gy of irradiation had a significantly lower average percentage of flap viability (56.9%) than animals receiving 23 Gy (90.9%) (P < .001). Furthermore, the longer duration until pedicle ligation after 40 Gy of irradiation led to significant increases in flap viability (P < .001 for analysis of variance). CONCLUSIONS: This animal model establishes that external beam irradiation at a total dose of 40 Gy leads to significantly delayed flap revascularization over time compared with 23-Gy irradiation. This model will allow future investigators to study novel therapies to improve healing and flap revascularization.

VEGF gene therapy augments localized angiogenesis and promotes anastomotic wound healing: a pilot study in a clinically relevant animal model.

BACKGROUND: Anastomotic leak related to ischemia is a source of significant morbidity and mortality in gastrointestinal surgery. The aim of this study was to apply growth factor gene transfection for the purpose of up-regulating angiogenesis, increasing anastomotic strength, and ultimately preventing dehiscence. METHODS: An opossum esophagogastrostomy model was employed. The human vascular endothelial growth factor (VEGF(165)) gene was incorporated into a recombinant plasmid. The VEGF plasmid vector was then complexed with a cationic synthetic carrier, polyethyleneimine. Control animals received plasmid devoid of VEGF(165) (n = 6). The experimental group received VEGF(165) plasmid (n = 5). After esophagogastrectomy and gastric tubularization, plasmid was injected into the submucosa of the neoesophagus at the anastomotic site. Conduit arteriography was performed before and 10 days after injection. Euthanasia occurred on post-injection day 10 and the anastomosis was removed en bloc. A second group of animals treated with VEGF(165) were euthanized 30 and 37 days post injection. Blood flow was measured with laser-Doppler prior to euthanasia. Ex vivo anastomotic bursting pressure was performed. Tissue samples were procured for RNA extraction and von Willebrand Factor staining. Microvessel counts were obtained by two blinded observers. Tissue VEGF transcript levels were measured with reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: There was one anastomotic leak in the control group. Experimental animals demonstrated significantly increased bursting pressure (104.25 +/- 6.2 vs 86.73 +/- 9.4 mmHg, p = 0.021) and neovascularization (33.87 +/- 9.6 vs 20.33 +/- 8.1 vessels/hpf, p = 0.032) compared to controls. In addition, there was a strongly positive correlation between the number of microvessels and bursting pressure (r = 0.808, p = 0.015, Pearson's). On angiographic examination, treated animals demonstrated more neovascularization compared to controls. RT-PCR demonstrated up to a 5.6-fold increase in VEGF mRNA in treated compared to controls. DISCUSSION: This description of gene therapy in gastrointestinal surgery using VEGF(165) transfection demonstrates increased angiogenesis with subsequently improved anastomotic healing in a clinically relevant model.

Revascularization of rat fasciocutaneous flap using CROSSEAL with VEGF protein or plasmid DNA expressing VEGF.

BACKGROUND: Fasciocutaneous tissue transfer is a common reconstructive procedure. Revascularization of flap tissue is an important component of tissue healing. Gene therapy offers an avenue through which the period of pedicle vascular dependency can be reduced. MATERIALS AND METHODS: Rat fasciocutaneous flaps were elevated and a two-hour ischemic time induced. Polycation complex (jet PEI) and human fibrin sealant CROSSEAL was applied between flap and underlying abdominal tissues. Group 1 (six rats) was the control; Group 2 (seven rats) had vascular endothelial growth factor (VEGF) protein applied; Group 3 (seven rats) had plasmid DNA expressing VEGF applied. Vascular pedicles were ligated on postoperative day 5, percentage flap survival evaluated on day 7. RESULTS: All flaps survived initial ischemia. Mean +/- SD percentage area of the flap that survived was 28.1 +/- 12.4 (Group 1), 71.6 +/- 16.2 (Group 2), and 77.5 +/- 12.7 (Group 3) (P < 0.001, Group 1-3, 2-3). No differences were observed between Groups 2 and 3. CONCLUSIONS: Locally administered VEGF protein or plasmid DNA expressing VEGF enhanced survival of fasciocutaneous flaps.