Biomimetic scaffold design for functional and integrative tendon repair.

Rotator cuff tears represent the most common shoulder injuries in the United States. The debilitating effect of this degenerative condition coupled with the high incidence of failure associated with existing graft choices underscores the clinical need for alternative grafting solutions. The 2 critical design criteria for the ideal tendon graft would require the graft to not only exhibit physiologically relevant mechanical properties but also be able to facilitate functional graft integration by promoting the regeneration of the native tendon-to-bone interface. Centered on these design goals, this review will highlight current approaches to functional and integrative tendon repair. In particular, the application of biomimetic design principles through the use of nanofiber- and nanocomposite-based scaffolds for tendon tissue engineering will be discussed. This review will begin with nanofiber-based approaches to functional tendon repair, followed by a section highlighting the exciting research on tendon-to-bone interface regeneration, with an emphasis on implementation of strategic biomimicry in nanofiber scaffold design and the concomitant formation of graded multi-tissue systems for integrative soft-tissue repair. This review will conclude with a summary and discussion of future directions.

The helix-loop-helix Id-1 inhibits PSA expression in prostate cancer cells.

The inhibitor of basic helix-loop-helix transcription factors, Id-1, is an important gene whose expression increases during prostate cancer progression and that upregulates proliferation, migration and invasion. We used microarray analysis to identify the downstream genes whose transcriptional expression is modulated by Id-1 protein. We compared gene expression in control LNCaP cells and Id-1-transduced LNCaP cells, which become significantly more aggressive after Id-1 overexpression, thus mimicking the high levels of Id-1 detected in metastatic cell lines. We used the Affy HTA U133A Expression Arrays with 45,000 probe sets representing more than 39,000 transcripts. We found that one of the most significantly downregulated genes on Id-1 expression was kallikrein 3 [also called prostate specific antigen (PSA)], the most commonly used biomarker of prostate cancer. Here, we show that the reduction in PSA mRNA and protein expression associated with high-grade prostate cancers, which generally express high levels of Id-1, could be the consequence of Id-1 overexpression.

Different real-time PCR systems yield different gene expression values.

Most polymerase chain reaction (PCR) systems employ pre-determined settings and proprietary master mixes that differ from one system to another. It is not known whether these differences may affect gene expression values. We compared two major real-time PCR technologies, from Life Technologies (formerly Applied Biosystems; ABI7500) and Roche Applied Science (LC480), using their default settings, proprietary reagents and other potential variables such as ramp rates and magnesium concentrations. We analyzed four genes (IL-8, COX2, ID-1 and CXCR2) in a human breast cancer cell line and found that two of them, though readily detected by ABI, were not detected using the Roche system. By altering some of the parameters and reagents used in the Roche protocol, we were able to detect expression of these two genes, but the level remained far below that detected by ABI, particularly for ID-1. When we tested three additional ID-1 primer pairs, two of these primer pairs yielded higher expression values in the LC system, yet still significantly lower than the values obtained in ABI. These results suggest critical differences in these two PCR systems, which could result in significant discrepancies in results reported by different laboratories.

Biomarkers correlate with colon cancer and risks: a preliminary study.

PURPOSE: We previously reported that gene expression analysis of biopsies of normal-appearing large intestinal mucosa can distinguish individuals with colonic cancer and many individuals at risk for colon cancer from controls. The purpose of this study was to determine whether noninvasively removed rectal swabs can identify individuals with colon cancer or risk of colon cancer as effectively as we previously demonstrated using biopsies. METHODS: Rectal mucosa cells were removed by rectal swabs, and their gene expression profiles were compared with those of biopsies removed by colonoscopy. Expression of 16 genes in the rectal mucosa of 12 individuals with colon cancer, 25 with polyps, 37 with family or self-reported cancer history, and 23 controls was measured by real-time reverse transcription-polymerase chain reaction. RESULTS: We found similar results using rectal swabs and biopsies. Groups of individuals with or at risk for cancer showed an altered gene expression profile compared with controls. Moreover, each of the 12 cancer patients showed altered expression relative to the mean of controls. CONCLUSIONS: Gene expression analysis using rectal swabs may provide a convenient way to screen for colon cancer.

Polymer fiber-based models of connective tissue repair and healing.

Physiologically relevant models of wound healing are essential for understanding the biology of connective tissue repair and healing. They can also be used to identify key cellular processes and matrix characteristics critical for the design of soft tissue grafts. Modeling the various stages of repair post tendon injury, polymer meshes of varying fiber diameter (nano-1 (390 nm) < nano-2 (740 nm) < micro (1420 nm)) were produced. Alignment was also introduced in the nano-2 group to model matrix undergoing biological healing rather than scar formation. The response of human tendon fibroblasts on these model substrates were evaluated over time as a function of fiber diameter and alignment. It was observed that the repair models of unaligned nanoscale fibers enhanced cell growth and collagen synthesis, while these outcomes were significantly reduced in the mature repair model consisting of unaligned micron-sized fibers. Organization of paxillin and actin on unaligned meshes was enhanced on micro- compared to nano-sized fibers, while the expression and activity of RhoA and Rac1 were greater on nanofibers. In contrast, aligned nanofibers promoted early cell organization, while reducing excessive cell growth and collagen production in the long term. These results show that the early-stage repair model of unaligned nanoscale fibers elicits a response characteristic of the proliferative phase of wound repair, while the more mature model consisting of unaligned micron-sized fibers is more representative of the remodeling phase by supporting cell organization while suppressing growth and biosynthesis. Interestingly, introduction of fiber alignment in the nanofiber model alters fibroblast response from repair to healing, implicating matrix alignment as a critical design factor for circumventing scar formation and promoting biological healing of soft tissue injuries.

Alteration of gene expression in macroscopically normal colonic mucosa from individuals with a family history of sporadic colon cancer.

PURPOSE: We have shown that the expression of several genes associated with human colon cancer is altered in the morphologically normal colonic mucosa (MNCM) of APC(min) mice and humans with colon cancers. To determine whether these alterations also occur in the MNCM of individuals who have not developed colon cancer but are at high risk of doing so, we measured gene expression in the MNCM of individuals with a family history of colon cancer. METHODS: Expression of 16 genes in the MNCM of 12 individuals with a first-degree relative with sporadic colon cancer and 16 normal controls were measured by quantitative reverse transcription-PCR. All subjects tested had normal colonoscopic examinations. Biopsy samples of MNCM were obtained from the ascending, transverse, descending, and rectosigmoid regions of the colon (2-8 biopsy samples were obtained from each region). RESULTS: Relative to normal controls, the expression of several genes, including PPAR-gamma, SAA1, and IL-8 were significantly altered in the macroscopically normal rectosigmoid mucosa from individuals with a family history of colon cancer. CONCLUSIONS: Molecular abnormalities that precede the appearance of adenomatous polyp are present in the MNCM of individuals who have a family history of colon cancer. This observation raises the possibility of screening for individuals who are at an increased risk of developing colon cancer by analysis of gene expression in rectosigmoid biopsy samples. To assess this possibility, prospective studies will be needed to determine whether or not altered gene expression is associated with the subsequent development of adenomatous polyps and/ or colonic carcinomas.

Alteration of gene expression in normal-appearing colon mucosa of APC(min) mice and human cancer patients.

The expression of many genes is altered in colon cancer, but the roles of these genes in carcinogenesis are unclear. Using real-time quantitative PCR, we demonstrated that several genes previously implicated in human colon cancer undergo altered expression in the APC(min) mouse adenomatous polyp, a precursor of cancer, as well as in normal-appearing surrounding mucosa. The five genes that were most highly up-regulated in mouse polyp were also significantly up-regulated in polyp-free colon mucosa. Similar changes occurred in morphologically normal mucosa of surgical sections taken from human cancer patients, frequently extending to the margins. Thus, morphologically normal colon mucosa in APC(min) mice and in human cancer patients is not metabolically normal. Altered gene expression in this tissue does not appear to result from a field effect because there was no correlation between extent of altered regulation and distance from polyp or tumor. Our data suggest that alterations of expression levels of these genes may be an early event in carcinogenesis and a marker of risk for the development of colon cancer.

Effect of neuroprotective drugs on gene expression in G93A/SOD1 mice.

Gene expression analysis is a powerful tool that has been used to define the pathological processes underlying many diseases. Several laboratories, including our own, have used this approach to identify molecular abnormalities in the G93A/SOD1 mouse, an animal model of amyotrophic lateral sclerosis (ALS). Here, we report the results of analysis of an expanded panel of genes throughout the entire lifetime in the spinal cord of these animals. In addition to upregulation of microglia/neuroinflammatory genes identified previously, we observed upregulation of metallothionein-I and -II (MT-I, MT-II). MT-I and MT-II play an important role in disposition of zinc ion, and other studies have also indicated their levels are altered in development of motor neuron disease in these animals. We also analyzed the effect on these expression profiles of several candidate drugs that have been shown to have neuroprotective effects in vivo or in vitro. That is, we asked whether administration to the G93A/SOD1 mice of any of these drugs could reverse the alterations in gene expression patterns that occur as the animals develop. The mice were given daily doses of these drugs when they were 9-11 weeks old, at a stage early in development of motor neuron disease, continuing for 5 weeks, at which time they were sacrificed. Treatment of the mice with l-carnosine, a dipeptide that scavenges free radicals and chelates zinc, did not affect expression of any of the genes altered in these animals. However, it did upregulate 3 genes unaffected by the presence of the G93A/SOD1 mutation: glial fibrillary acidic protein (GFAP), stroma-derived factor-1 (SDF-1), and excitatory amino acid transporter-2 (EAAT2). In contrast, metallothionein-III (MT-III) was downregulated. Treatment of the animals with baicalein, an herbal extract with anti-inflammatory and numerous other effects, downregulated the microglia markers CD68, CD80, and CD86, all of which were upregulated in untreated mutant animals. Baicalein treatment also downregulated tumor necrosis factor receptor (TNFRp55) and upregulated noninducible nitric oxide synthase (nNOS) and glutamine synthase (GS). These 3 genes were unaffected by the presence of the G93A mutation. We discuss the implication of these results for testing the effects of these and other candidate drugs in mutant SOD1 mice.

[Dmt(1)]DALDA is highly selective and potent at mu opioid receptors, but is not cross-tolerant with systemic morphine.

The clinical effectiveness of morphine is limited by several side effects, including the development of tolerance and dependence. Most of these side effects are believed to be mediated by central opioid receptors; therefore, hydrophilic opioids, which don't cross the blood-brain barrier, may have advantages over morphine in some clinical applications. We recently synthesized several analogues of DALDA (Tyr-D-Arg-Phe-Lys-NH2), a highly hydrophilic peptide derived from the endogenous opioid peptide dermorphin; all of them, particularly [Dmt(1)] DALDA (Dmt - 2',6'-dimethyl tyrosine), had high potency and selectivity at mu receptors, the target of morphine, in activity assays. Here we report the pharmacological characterization of [Dmt(1)] DALDA in the whole animal. [Dmt(1)]DALDA was 40 times more potent than morphine in inducing antinociception in mice when both drugs were given s.c., and 6-14 times more potent than DAMGO, a selective m agonist, when both drugs were given it. However, [Dmt(1)]DALDA showed poor cross-tolerance to morphine; thus chronic morphine treatment of animals increased the antinociceptive AD(50) of systemic [Dmt(1)]DALDA two fold or less, as compared to an 8-9-fold increase for morphine and a 4-5-fold increase for DAMGO. The antinociceptive activity of [Dmt(1)]DALDA (i.t) was blocked by CTAP, a selective mu antagonist, but not by TIPP psi, a selective delta antagonist, nor by nor-BNI, a selective kappa antagonist. [Dmt(1)]DALDA-induced antinociception was also blocked by naloxone methiodide, an antagonist that does not cross the blood-brain barrier, when agonist and antagonist were given i.t. or i.c.v., but not when they were given s.c. We conclude that [Dmt(1)] DALDA is a highly potent analgesic acting at mu receptors. Though it appears to penetrate the blood-brain barrier, it exhibits low cross-tolerance to morphine, suggesting that it may have advantages over the latter in certain clinical applications.

Tolerance develops in spinal cord, but not in brain with chronic [Dmt1]DALDA treatment.

Previously, we reported that H-2',6'-dimethyltyrosine [Dmt(1)]-d-Arg-Phe-Lys-NH(2) (DALDA), an analogue of the naturally occurring opioid peptide dermorphin, is a highly potent and selective mu receptor agonist with low cross-tolerance to morphine. In the present study, we investigated the effect of treating mice chronically with [Dmt(1)]DALDA. The AD(50) of [Dmt(1)]DALDA (s.c.) increased eight-fold in animals given this drug chronically; in contrast, the AD(50) increased two-fold in mice chronically treated with morphine. The AD(50) of morphine (s.c.) in these [Dmt(1)]DALDA-treated animals was increased more than 120 times, while that of the more selective mu agonist [d-Ala(2)-MePhe(4)-Gly-ol(5)]enkephalin (DAMGO) given intrathecally was increased more than 240 times. However, the AD(50) of DAMGO given intracerebroventricularly was essentially the same in animals treated chronically with [Dmt(1)]DALDA as in naive animals. The dose of naloxone required to precipitate withdrawal in [Dmt(1)]DALDA-treated animals was 20 times lower than that in morphine-tolerant animals. Using real-time quantitative PCR, we found that expression of the mu opioid receptor, delta opioid receptor, preproenkephalin and preprodynorphin genes was upregulated in the brain by [Dmt(1)]DALDA treatment. No significant changes in expression of opioid receptor or opioid peptide genes were detected in the spinal cord of [Dmt(1)]DALDA-treated mice, nor in the brain or spinal cord of morphine-treated mice. We conclude that a high degree of tolerance to [Dmt(1)]DALDA develops in the spinal cord but not brain, and cannot be accounted for by changes in expression of opioid receptors or opioid peptides in these tissues.

Opioid receptor polymorphisms and opioid abuse.

The sequencing of the human genome is only the first step. The next step is to determine the function of these genes and in particular, how alterations in specific genes lead to major human disorders. Many laboratories are now focusing on identifying and characterizing single nucleotide polymorphisms (SNPs), to determine which correlate in frequency with certain population groups who may be particularly susceptible to certain diseases. The mu opioid receptor (MOR), which mediates the clinically important analgesic effects of drugs like morphine as well as the euphoria sought by heroin abusers, exhibits several dozen polymorphisms. Several of these are associated with altered receptor function and individuals at risk for drug abuse.

Opioid receptor interactions: local and nonlocal, symmetric and asymmetric, physical and functional.

The pharmacological effects of opioid drugs and endogenous opioid peptides are mediated by several kinds of receptors, the major ones being mu, delta and kappa. Though classically it has been thought that a particular effect mediated by a drug or other ligand results from its interaction with a single type of receptor, accumulating evidence demonstrates that interactions between receptors play a major role in opioid actions. These interactions may be either local, involving receptors within the same tissue, or nonlocal, between receptors located in different tissues. Nonlocal interactions always involve intercellular mechanisms, whereas local interactions may involve either intercellular or intracellular interactions, the latter including physical association of receptors. Both local and nonlocal interactions, moreover, may be either symmetric, with ligand interaction at one receptor affecting interaction at the other, or asymmetric; and either potentiating or inhibitory. In this article we discuss major examples of these kinds of interactions, and their role in the acute and chronic effects of opioids. Knowledge of these interactions may have important implications for the design of opioids with more specific actions, and for eliminating the addictive liability of these drugs.

Towards universal screening for colon cancer: a cheap, reliable, noninvasive test using gene expression analysis of rectal swabs.

Though colon cancer is the second leading cause of cancer deaths in the US, it is entirely preventable through early screening to detect and remove adenomatous polyps. Colonoscopy has long been regarded as the "gold standard" but is expensive, invasive, and uncomfortable, and only about half those considered at risk for colon cancer currently submit to colonoscopy or to less reliable alternatives such as fecal occult blood test. Here we describe the use of gene expression analysis to detect altered expression of certain genes associated with not only colon cancer but also polyps. The analysis can be performed on rectal swabs, with specimens provided in a routine doctor's office visit. The existence of this cheap and simple test, together with an active program to encourage individuals to submit to screening, could help eradicate colon cancer.

Role of zinc in ALS.

The causes of amyotrophic lateral sclerosis (ALS) are poorly understood. A small proportion, about 2%, is associated with a mutation in the superoxide dismutase (SOD1) gene, and mice expressing this mutant gene exhibit a progressive, ALS-like neurodegenerative disease. Studies of these animals, as well as of human post mortem tissue, reveal the presence of multiple pathological processes, including oxidative stress, glutamate excitotoxicity, neuroinflammation, mitochondrial degeneration, alterations in neurofilaments and neurotubules, mitochondrial damage, aggregation of proteins, abnormalities in growth factors, and apoptosis. We propose that alterations in the disposition of zinc ions may be important in the initiation and development of ALS. SOD1 binds zinc, and many of the mutant forms of this enzyme associated with ALS show altered zinc binding. Alterations in the expression of metallothioneins (MTs), which regulate cellular levels of zinc, have been reported in mutant SOD1 mice, and deletion of MTs in these animals accelerates disease progression. Zinc plays a key role in all the pathological processes associated with ALS. Our zinc hypothesis also may help explain evidence for environmental factors in some cases of ALS, such as in the Chamorro tribe in Guam and in the Gulf War.

Altered gene expression in normal colonic mucosa of individuals with polyps of the colon.

PURPOSE: Expression levels of many genes are altered in colon cancer, relative to normal colonic mucosa. We recently reported that such differences also exist between grossly normal colonic mucosa of individuals with and without colon cancer, and between individuals with and without a family history of colon cancer. Here we report a study of individuals with no cancer but with polyps in the transverse, ascending/descending, or rectosigmoid colon. METHODS: Biopsies of grossly normal-appearing colonic mucosa from the rectosigmoid colon were taken from individuals with polyps, with or without personal/family history of colon cancer, and gene expression profiles compared with those from biopsies of control patients, with no polyps or known personal/family history. A global expression analysis was conducted of the same 15 genes used in our previous studies. RESULTS: We found significant differences in gene expression in normal-appearing rectosigmoid colonic mucosa between individuals with polyps and controls, regardless of whether personal or family history of cancer was present. CONCLUSIONS: Alterations in gene expression patterns in morphologically normal-appearing colonic mucosa are associated with the presence of adenomatous polyps. Prospective studies will be required to determine whether these alterations in gene expression can be used to predict risk of developing colon cancer.

Morphine tolerance in spinal cord is due to interaction between mu- and delta-receptors.

When the opioid agonist morphine is given chronically and systemically to mice by pellet implantation for 3 days, the animals develop substantial tolerance to the antinociceptive effect of a test dose of morphine given systemically. When the test dose is administered to the spinal cord, however, very little tolerance is observed. We tested six strains of mice differing in the degree to which they develop systemic tolerance to morphine and found that none of them developed significant tolerance to spinal morphine. However, most of these strains did develop substantial spinal tolerance to antinociception induced by the selective mu-agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and by the selective delta-agonist [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE). Moreover, in naïve animals, the antinociceptive effect of both DAMGO and DPDPE was blocked by D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2), a selective mu-antagonist, indicating that both agonists mediate antinociception in the spinal cord through mu-receptors. In addition to directly mediating antinociception, however, DPDPE potentiated the antinociceptive activity of DAMGO in the spinal cord of naïve animals, and this antinociception was blocked by the delta-antagonist H-TyrTicPsi[CH(2)NH]Phe-Thr-OH (TIPPpsi), indicating mediation through delta-receptors. In contrast, in tolerant animals, TIPPpsi enhanced the antinociception of DAMGO. These results thus demonstrate not only that mu- and delta-opioid receptors interact in naïve animals, but that the nature of this interaction changes during tolerance, from a potentiation to an inhibition. The lack of tolerance to spinal morphine may result from the ability of morphine to act as a partial antagonist at delta-receptors.

Id-1 as a molecular target in therapy for breast cancer cell invasion and metastasis.

Mammary epithelial cells constitutively expressing Id-1 protein are unable to differentiate, acquire the ability to proliferate, and invade the extracellular matrix. In addition, Id-1 is aberrantly over-expressed in aggressive and metastatic breast cancer cells, as well as in human breast tumor biopsies from infiltrating carcinomas, suggesting Id-1 might be an important regulator of breast cancer progression. We show that human metastatic breast cancer cells become significantly less invasive in vitro and less metastatic in vivo when Id-1 is down-regulated by stable transduction with antisense Id-1. Expression of the matrix metalloproteinase MT1-MMP is decreased in proportion to the decrease in Id-1 protein levels, representing a potential mechanism for the reduction of invasiveness. Further, to more accurately recapitulate the biology of and potential therapeutic approaches to tumor metastasis, we targeted Id-1 expression systemically in tumor-bearing mice by using a nonviral approach. We demonstrate significant reduction of both Id-1 and MT1-MMP expressions as well as the metastatic spread of 4T1 breast cancer cells in syngeneic BALB/c mice. In conclusion, our studies have identified Id-1 as a critical regulator of breast cancer progression and suggest the feasibility of developing novel therapeutic approaches to target Id-1 expression to reduce breast cancer metastasis in humans.