Etiopathogenesis of adolescent idiopathic scoliosis: Review of the literature and new epigenetic hypothesis on altered neural crest cells migration in early embryogenesis as the key event.

Adolescent idiopathic scoliosis (AIS) affects 2-3% of children. Numerous hypotheses on etiologic/causal factors of AIS were investigated, but all failed to identify therapeutic targets and hence failed to offer a cure. Therefore, currently there are only two options to minimize morbidity of the patients suffering AIS: bracing and spinal surgery. From the beginning of 1960th, spinal surgery, both fusion and rod placement, became the standard of management for progressive adolescent idiopathic spine deformity. However, spinal surgery is often associated with complications. These circumstances motivate AIS scientific community to continue the search for new etiologic and causal factors of AIS. While the role of the genetic factors in AIS pathogenesis was investigated intensively and universally recognized, these studies failed to nominate mutation of a particular gene or genes combination responsible for AIS development. More recently epigenetic factors were suggested to play causal role in AIS pathogenesis. Sharing this new approach, we investigated scoliotic vertebral growth plates removed during vertebral fusion (anterior surgery) for AIS correction. In recent publications we showed that cells from the convex side of human scoliotic deformities undergo normal chondrogenic/osteogenic differentiation, while cells from the concave side acquire a neuronal phenotype. Based on these facts we hypothesized that altered neural crest cell migration in early embryogenesis can be the etiological factor of AIS. In particular, we suggested that neural crest cells failed to migrate through the anterior half of somites and became deposited in sclerotome, which in turn produced chondrogenic/osteogenic-insufficient vertebral growth plates. To test this hypothesis we conducted experiments on chicken embryos with arrest neural crest cell migration by inhibiting expression of Paired-box 3 (Pax3) gene, a known enhancer and promoter of neural crest cells migration and differentiation. The results showed that chicken embryos treated with Pax3 siRNA (microinjection into the neural tube, 44 h post-fertilization) progressively developed scoliotic deformity during maturation. Therefore, this analysis suggests that although adolescent idiopathic scoliosis manifests in children around puberty, the real onset of the disease is of epigenetic nature and takes place in early embryogenesis and involves altered neural crest cells migration. If these results confirmed and further elaborated, the hypothesis may shed new light on the etiology and pathogenesis of AIS.

Pattern of organ remodeling in chronic non-communicable diseases is due to endogenous regulations and falls under the category of Kauffman's self-organization: A case of arterial neointimal pathology.

Clinical diagnosis is based on analysis of pathologic findings that may result in perceived patterns. The same is true for diagnostic pathology: Pattern analysis is a foundation of the histopathology-based diagnostic system and, in conjunction with clinical and laboratory findings, forms a basis for the classification of diseases. Any histopathology diagnosis is based on the explicit assumption that the same diseased condition should result in formation of the same (or highly similar) morphologic patterns in different individuals; it is a standard approach in microscopic pathology, including that of non-communicable chronic diseases with organ remodeling. During fifty years of examining diseased tissues under microscopy, I keep asking the same question: Why is a similarity of patterns expected for chronic organ remodeling? For infection diseases, xenobiotic toxicity and deficiencies forming an identical pathologic pattern in different individuals is understandable and logical: The same infection, xenobiotic, or deficiency strikes the same target, which results in identical pathology. The same is true for Mendelian diseases: The same mutations lead to the same altered gene expressions and the same pathologic pattern. But why does this regularity hold true for chronic diseases with organ remodeling? Presumable causes (or risk factors) for a particular chronic disease differ in magnitude and duration between individuals, which should result in various series of transformations. Yet, mysteriously enough, pathological remodeling in a particular chronic disease always falls into a main dominating pattern, perpetuating and progressing in a similar fashion in different patients. Furthermore, some chronic diseases of different etiologies and dissimilar causes/risk factors manifest as identical or highly similar patterns of pathologic remodeling. HYPOTHESIS: I hypothesize that regulations governing a particular organ's chronic remodeling were selected in evolution as the safest response to various insults and physiologic stress conditions. This hypothesis implies that regulations directing diseased chronic remodeling always preexist but normally are controlled; this control can be disrupted by a diverse range of non-specific signals, liberating the pathway for identical pathologic remodeling. This hypothesis was tested in an analysis of arterial neointimal formation, the identical pathology occurring in different diseases and pathological conditions: graft vascular disease in organ transplantation, in-stent restenosis, peripheral arterial diseases, idiopathic intimal hyperplasia, Kawasaki disease, coronary atherosclerosis and as reaction to drugs. The hypothesis suggests that arterial intimal cells are poised between only two alternative pathways: the pathway with controlled intimal cell proliferation or the pathway where such control is disrupted, ultimately leading to the progressive neointimal pathology. By this property the arterial neointimal formation constitutes a special case of Kauffman's self-organization. This new hypothesis gives a parsimonious explanation for identical pathological patterns of arterial remodeling (neointimal formation), which occurs in diseases of different etiologies and due to dissimilar causes/risk factors, or without any etiology and causes/risk factors at all. This new hypothesis also suggests that regulation facilitating intimal cell proliferation cannot be overwritten or annulled because this feature is vital for arterial differentiation, cell renewal, and integrity. This hypothesis suggests that studying numerous, and likely interchangeable, non-specific signals that disrupt regulation controlling intimal cell proliferation is unproductive; instead, a study of the controlling regulation(s) itself should be a priority of our research.

Development of an RNAi therapeutic for alpha-1-antitrypsin liver disease.

The autosomal codominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria, and inflammation, leading to fibrosis, cirrhosis, and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first-generation intravenously administered RNA interference (RNAi) therapeutic ARC-AAT and then with next-generation subcutaneously administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis, and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria, and autophagosomes in hepatocytes was evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation, and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in phase II/III clinical trials.

A hypothesis on paradoxical privileged portal vein metastasis of hepatocellular carcinoma. Can organ evolution shed light on patterns of human pathology, and vice versa?

Unlike other carcinomas, hepatocellular carcinoma (HCC) metastasizes to distant organs relatively rarely. In contrast, it routinely metastasizes to liver vasculature/liver, affecting portal veins 3-10 times more often than hepatic veins. This portal metastatic predominance is traditionally rationalized within the model of a reverse portal flow, due to accompanying liver cirrhosis. However, this intuitive model is not coherent with facts: 1) reverse portal flow occurs in fewer than 10% of cirrhotic patients, while portal metastasis occurs in 30-100% of HCC cases, and 2) portal vein prevalence of HCC metastasis is also characteristic of HCC in non-cirrhotic livers. Therefore, we must assume that the route for HCC metastatic dissemination is the same as for other carcinomas: systemic dissemination via the draining vessel, i.e., via the hepatic vein. In this light, portal prevalence versus hepatic vein of HCC metastasis appears as a puzzling pattern, particularly in cases when portal HCC metastases have appeared as the sole manifestation of HCC. Considering that other GI carcinomas (colorectal, pancreatic, gastric and small bowel) invariably disseminate via portal vein, but very rarely form portal metastasis, portal prevalence of HCC metastasis appears as a paradox. However, nature does not contradict itself; it is rather our wrong assumptions that create paradoxes. The 'portal paradox' becomes a logical event within the hypothesis that the formation of the unique portal venous system preceded the appearance of liver in evolution of chordates. The analysis suggests that the appearance of the portal venous system, supplying hormones and growth factors of pancreatic family, which includes insulin, glucagon, somatostatin, and pancreatic polypeptide (HGFPF) to midgut diverticulum in the early evolution of chordates (in an Amphioxus-like ancestral animal), promoted differentiation of enterocytes into hepatocytes and their further evolution to the liver of vertebrates. These promotional-dependent interactions are conserved in the vertebrate lineage. I hypothesize that selective homing and proliferation of malignant hepatocytes (i.e., HCC cells) in the portal vein environment are due to a uniquely high concentration of HGFPF in portal blood. HGFPF are also necessary for liver function and renewal and are significantly extracted by hepatocytes from passing blood, creating a concentration gradient of HGFPF between the portal blood and hepatic vein outflow, making post-liver vasculature and remote organs less favorable spaces for HCC growth. It also suggested that the portal vein environment (i.e., HGFPF) promotes the differentiation of more aggressive HCC clones from already-seeded portal metastases, explaining the worse outcome of HCC with the portal metastatic pattern. The analysis also offers new hypothesis on the phylogenetic origin of the hepatic diverticulum of cephalochordates, with certain implications for the modeling of the chordate phylogeny.

A New Look at Causal Factors of Idiopathic Scoliosis: Altered Expression of Genes Controlling Chondroitin Sulfate Sulfation and Corresponding Changes in Protein Synthesis in Vertebral Body Growth Plates.

Background: In a previous report, we demonstrated the presence of cells with a neural/glial phenotype on the concave side of the vertebral body growth plate in Idiopathic Scoliosis (IS) and proposed this phenotype alteration as the main etiological factor of IS. In the present study, we utilized the same specimens of vertebral body growth plates removed during surgery for Grade III-IV IS to analyse gene expression. We suggested that phenotype changes observed on the concave side of the vertebral body growth plate can be associated with altered expression of particular genes, which in turn compromise mechanical properties of the concave side. Methods: We used a Real-Time SYBR Green PCR assay to investigate gene expression in vertebral body growth plates removed during surgery for Grade III-IV IS; cartilage tissues from human fetal spine were used as a surrogate control. Special attention was given to genes responsible for growth regulation, chondrocyte differentiation, matrix synthesis, sulfation and transmembrane transport of sulfates. We performed morphological, histochemical, biochemical, and ultrastructural analysis of vertebral body growth plates. Results: Expression of genes that control chondroitin sulfate sulfation and corresponding protein synthesis was significantly lower in scoliotic specimens compared to controls. Biochemical analysis showed 1) a decrease in diffused proteoglycans in the total pool of proteoglycans; 2) a reduced level of their sulfation; 3) a reduction in the amount of chondroitin sulfate coinciding with raising the amount of keratan sulfate; and 4) reduced levels of sulfation on the concave side of the scoliotic deformity. Conclusion: The results suggested that altered expression of genes that control chondroitin sulfate sulfation and corresponding changes in protein synthesis on the concave side of vertebral body growth plates could be causal agents of the scoliotic deformity.

A New Look at Etiological Factors of Idiopathic Scoliosis: Neural Crest Cells.

Idiopathic scoliosis is one of the most common disabling pathologies of children and adolescents. Etiology and pathogenesis of idiopathic scoliosis remain unknown. To study the etiology of this disease we identified the cells' phenotypes in the vertebral body growth plates in patients with idiopathic scoliosis. Materials and methods: The cells were isolated from vertebral body growth plates of the convex and concave sides of the deformity harvested intraoperatively in 50 patients with scoliosis. Cells were cultured and identified by methods of common morphology, neuromorphology, electron microscopy, immunohistochemistry and PCR analysis. Results: Cultured cells of convex side of deformation were identified as chondroblasts. Cells isolated from the growth plates of the concave side of the deformation showed numerous features of neuro- and glioblasts. These cells formed synapses, contain neurofilaments, and expressed neural and glial proteins. Conclusion: For the first time we demonstrated the presence of cells with neural/glial phenotype in the concave side of the vertebral body growth plate in scoliotic deformity. We hypothesized that neural and glial cells observed in the growth plates of the vertebral bodies represent derivatives of neural crest cells deposited in somites due to alterations in their migratory pathway during embryogenesis. We also propose that ectopic localization of cells derived from neural crest in the growth plate of the vertebral bodies is the main etiological factor of the scoliotic disease.

Privileged portal metastasis of hepatocellular carcinoma in light of the coevolution of a visceral portal system and liver in the chordate lineage: a search for therapeutic targets.

Hepatocellular carcinoma (HCC) disseminates systemically, but metastases occur in distant organs only in minority of patients, whereas HCC routinely metastasizes to liver and its vessels. HCC cells disseminate via hepatic veins, but portal veins are affected by metastasis more frequently than are hepatic veins, and correlates with poor prognosis. In this review, I suggest that privileged HCC portal metastasis occurs because of high levels of pancreatic family hormones and growth factors (PHGFs) in the portal blood. The analysis suggests that the appearance of the portal system carrying PHGFs in the evolution of invertebrate chordate (Amphioxus) led to the evolution of the liver in vertebrate; given that the portal pattern of HCC metastasis and selection of more-aggressive clones are PHGF dependent, PHGFs and their ligands constitute therapeutic targets.

Excessive intimal hyperplasia in human coronary arteries before intimal lipid depositions is the initiation of coronary atherosclerosis and constitutes a therapeutic target.

The consensus hypothesis on coronary atherosclerosis suggests high LDL-C levels as the major cause and pursues it as the therapeutic target, explicitly assuming: (i) tunica intima of human coronaries consists of only one cell layer - endothelium, situated on a thin layer of scarcely cellular matrix; and (ii) subendothelial lipoprotein retention initiates the disease. Facts showed: (i) normal tunica intima invariably consists of multiple cellular layers; and (ii) initial lipid depositions occurred in the deepest layers of tunica intima. This review suggests that coronary atherosclerosis starts with pathological intimal expansion, resulting in intimal hypoxia and neovascularization from adventitial vasa vasorum, facilitating lipoprotein extraction by previously avascular deep intimal tissues. Until the hypothesis incorporates real knowledge, our efforts will probably be off-target.

Loco-regional cancer drug therapy: present approaches and rapidly reversible hydrophobization (RRH) of therapeutic agents as the future direction.

Insufficient drug uptake by solid tumors remains the major problem for systemic chemotherapy. Many studies have demonstrated anticancer drug effects to be dose-dependent, although dose-escalation studies have resulted in limited survival benefit with increased systemic toxicities. One solution to this has been the idea of loco-regional drug treatments, which offer dramatically higher drug concentrations in tumor tissues while minimizing systemic toxicity. Although loco-regional delivery has been most prominent in cancers of the liver, soft tissues and serosal peritoneal malignancies, survival benefits are very far from desirable. This review discusses the evolution of loco-regional treatments, the present approaches and offers rapidly reversible hydrophobization of drugs as the new future direction.

Dendritic cell-based cancer immunotherapy: the stagnant approach and a theoretical solution.

Anticancer dendritic cells (DC) therapy currently uses in vitro propagation of the patient's DC and pulsing with tumor antigens. However, clinical achievements are far from desirable. Here, I suggest that the lack of anticipated responses could be because cancer cells continuously mutate, whereas the population of tumor antigens from the excised tumor is genetically static, and because there is an absence of biologic mechanisms to facilitate intratumoral DC retention, which is needed for DC pulsing. I hypothesize that stable tumor transfection with fetal liver tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) DNAs will induce homing, propagation and maturation of intratumoral DC. This must be followed by drug-induced apoptosis of tumor cells, to ensure the release of tumor antigens for DC pulsing. Then, regardless of any mutation of tumor cells, they would always incite DC propagation and maturation, pulsing and antitumor immunity.

Treg cells in pancreatic lymph nodes: the possible role in diabetogenesis and ß cell regeneration in a T1D model.

Previously, we established a model in which physiologically adequate function of the autologous ß cells was recovered in non-obese diabetic (NOD) mice after the onset of hyperglycemia by rendering them hemopoietic chimera. These mice were termed antea-diabetic. In the current study, we addressed the role of T regulatory (Treg) cells in the mechanisms mediating the restoration of euglycemia in the antea-diabetic NOD model. The data generated in this study demonstrated that the numbers of Treg cells were decreased in unmanipulated NOD mice, with the most profound deficiency detected in the pancreatic lymph nodes (PLNs). The impaired retention of the Treg cells in the PLNs correlated with the locally compromised profile of the chemokines involved in their trafficking, with the most prominent decrease observed in SDF-1. The amelioration of autoimmunity and restoration of euglycemia observed in the antea-diabetic mice was associated with restoration of the Treg cell population in the PLNs. These data indicate that the function of the SDF-1/CXCR4 axis and the retention of Treg cells in the PLNs have a potential role in diabetogenesis and in the amelioration of autoimmunity and ß cell regeneration in the antea-diabetic model. We have demonstrated in the antea-diabetic mouse model that lifelong recovery of the ß cells has a strong correlation with normalization of the Treg cell population in the PLNs. This finding offers new opportunities for testing the immunomodulatory regimens that promote accumulation of Treg cells in the PLNs as a therapeutic approach for type 1 diabetes (T1D).

Neovascularization of coronary tunica intima (DIT) is the cause of coronary atherosclerosis. Lipoproteins invade coronary intima via neovascularization from adventitial vasa vasorum, but not from the arterial lumen: a hypothesis.

BACKGROUND: An accepted hypothesis states that coronary atherosclerosis (CA) is initiated by endothelial dysfunction due to inflammation and high levels of LDL-C, followed by deposition of lipids and macrophages from the luminal blood into the arterial intima, resulting in plaque formation. The success of statins in preventing CA promised much for extended protection and effective therapeutics. However, stalled progress in pharmaceutical treatment gives a good reason to review logical properties of the hypothesis underlining our efforts, and to reconsider whether our perception of CA is consistent with facts about the normal and diseased coronary artery. ANALYSIS: To begin with, it must be noted that the normal coronary intima is not a single-layer endothelium covering a thin acellular compartment, as claimed in most publications, but always appears as a multi-layer cellular compartment, or diffuse intimal thickening (DIT), in which cells are arranged in many layers. If low density lipoprotein cholesterol (LDL-C) invades the DIT from the coronary lumen, the initial depositions ought to be most proximal to blood, i.e. in the inner DIT. The facts show that the opposite is true, and lipids are initially deposited in the outer DIT. This contradiction is resolved by observing that the normal DIT is always avascular, receiving nutrients by diffusion from the lumen, whereas in CA the outer DIT is always neovascularized from adventitial vasa vasorum. The proteoglycan biglycan, confined to the outer DIT in both normal and diseased coronary arteries, has high binding capacity for LDL-C. However, the normal DIT is avascular and biglycan-LDL-C interactions are prevented by diffusion distance and LDL-C size (20 nm), whereas in CA, biglycan in the outer DIT can extract lipoproteins by direct contact with the blood. These facts lead to the single simplest explanation of all observations: (1) lipid deposition is initially localized in the outer DIT; (2) CA often develops at high blood LDL-C levels; (3) apparent CA can develop at lowered blood LDL-C levels. This mechanism is not unique to the coronary artery: for instance, the normally avascular cornea accumulates lipoproteins after neovascularization, resulting in lipid keratopathy. HYPOTHESIS: Neovascularization of the normally avascular coronary DIT by permeable vasculature from the adventitial vasa vasorum is the cause of LDL deposition and CA. DIT enlargement, seen in early CA and aging, causes hypoxia of the outer DIT and induces neovascularization. According to this alternative proposal, coronary atherosclerosis is not related to inflammation and can occur in individuals with normal circulating levels of LDL, consistent with research findings.

Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 µg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

The acquisition of narrow binding specificity by polyspecific natural IgM antibodies in a semi-physiological environment.

Natural IgM antibodies (Abs) play an important role in clearing pathogens, enhancing immune responses, and preventing autoimmunity. However, the molecular mechanisms that mediate the functions of natural IgM Abs are understood only to a limited degree. This shortcoming is largely due to the fact that isolated natural IgM Abs are commonly polyspecific and recognize a variety of antigens (Ags) with no apparent structural homology. It is generally believed that polyspecificity is an inherent property of natural Abs. However, there is increasing evidence that polyspecificity may be induced by mild denaturing conditions. In this study, we compared the specificity of three polyspecific IgM Abs in conventional buffers and undiluted sera deficient in immunoglobulins. All three Abs lost their polyspecificity in serum. They no longer reacted with conventional screening Ags, including hapten-BSA conjugates, ssDNA, thyroglobulin and myosin, but fully retained their reactivity with cognate peptide Ags selected from a T7 phage library. The acquisition of narrow specificity by polyspecific IgM in serum was also observed with muscle tissue sections used as a source of endogenous Ags. The loss of polyspecificity by different Abs was apparently dependent on the presence of different serum constituents. The results of this study suggest that the seemingly inherent polyspecificity of many natural IgM Abs may be largely an in vitro phenomenon related to the lack of normal serum components in the medium. Potential mechanisms underlying the loss of polyreactivity are discussed.

Analysis of arterial intimal hyperplasia: review and hypothesis.

BACKGROUND: Despite a prodigious investment of funds, we cannot treat or prevent arteriosclerosis and restenosis, particularly its major pathology, arterial intimal hyperplasia. A cornerstone question lies behind all approaches to the disease: what causes the pathology? HYPOTHESIS: I argue that the question itself is misplaced because it implies that intimal hyperplasia is a novel pathological phenomenon caused by new mechanisms. A simple inquiry into arterial morphology shows the opposite is true. The normal multi-layer cellular organization of the tunica intima is identical to that of diseased hyperplasia; it is the standard arterial system design in all placentals at least as large as rabbits, including humans. Formed initially as one-layer endothelium lining, this phenotype can either be maintained or differentiate into a normal multi-layer cellular lining, so striking in its resemblance to diseased hyperplasia that we have to name it "benign intimal hyperplasia". However, normal or "benign" intimal hyperplasia, although microscopically identical to pathology, is a controllable phenotype that rarely compromises blood supply. It is remarkable that each human heart has coronary arteries in which a single-layer endothelium differentiates early in life to form a multi-layer intimal hyperplasia and then continues to self-renew in a controlled manner throughout life, relatively rarely compromising the blood supply to the heart, causing complications requiring intervention only in a small fraction of the population, while all humans are carriers of benign hyperplasia. Unfortunately, this fundamental fact has not been widely appreciated in arteriosclerosis research and medical education, which continue to operate on the assumption that the normal arterial intima is always an "ideal" single-layer endothelium. As a result, the disease is perceived and studied as a new pathological event caused by new mechanisms. The discovery that normal coronary arteries are morphologically indistinguishable from deadly coronary arteriosclerosis continues to elicit surprise. CONCLUSION: Two questions should inform the priorities of our research: (1) what controls switch the single cell-layer intimal phenotype into normal hyperplasia? (2) how is normal (benign) hyperplasia maintained? We would be hard-pressed to gain practical insights without scrutinizing our premises.

Rapidly reversible hydrophobization: an approach to high first-pass drug extraction.

We have investigated a rapidly reversible hydrophobization of therapeutic agents for improving first-pass uptake in locoregional drug therapy. This approach involves the attachment of a hydrophobic moiety to the drug by highly labile chemical linkages that rapidly hydrolyze upon injection. Hydrophobization drastically enhances cell-membrane association of the prodrug and, consequently, drug uptake, while the rapid lability protects nontargeted tissues from exposure to the highly active agent. Using the membrane-impermeable DNA intercalator propidium iodide, and melphalan, we report results from in vitro cellular internalization and toxicity studies. Additionally, we report in vivo results after a single liver arterial bolus injection, demonstrating both tumor targeting and increased survival in a mouse tumor model.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules.

BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. II. Morphological studies.

BACKGROUND: The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA. METHODS: We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance. RESULTS: Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them. CONCLUSIONS: Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression.

Small proline-rich proteins 2 are noncoordinately upregulated by IL-6/STAT3 signaling after bile duct ligation.

Small proline-rich proteins 2 (SPRR2) are coordinately expressed with other epidermal differential complex (EDC) genes in the skin. They function as crosslinking proteins that form bridges between other proteins that comprise the cornified cell envelope, which is the major barrier against the environment. IL-6 is invariably produced at sites of biliary tract injury and IL-6-deficient (IL-6(-/-)) mice show impaired barrier function after bile duct ligation (BDL). Screening microarray analysis identified noncoordinate expression of SPRR2 as a candidate gene that is: (a) expressed in biliary epithelial cells (BEC); (b) IL-6 responsive; and (c) potentially related to biliary barrier function. Therefore, we studied in detail the regulation of BEC SPRR2A expression, in vitro; and tested the hypothesis that if BEC SPRR2 expression contributes to biliary barrier function, it should be increased after BDL in IL-6-wild type (IL-6(+/+)) mice and not in IL-6(-/-) mice. In vitro studies confirmed that IL-6/gp130-signaling, mediated primarily by signal transducer and activator of transcription 3 (STAT3), stimulated noncoordinate BEC SPRR2 expression. In vivo, noncoordinate upregulation of BEC SPRR2 expression after BDL was seen in the IL-6(+/+) mice and was unrelated to squamous metaplasia. IL-6(-/-) mice showed deficient BEC SPRR2 expression after BDL associated with impaired barrier function, as evidenced by smaller diameters of obstructed ducts, decreased bile volume, and an inability to form 'white bile' compared to IL-6(+/+) mice at 12 weeks after BDL. IL-6 replacement therapy reversed the barrier defect in IL-6(-/-) mice after BDL, coincident with recovery of SPRR2A expression. SPRR2 in diseased mouse and human liver localized subjacent to the apical plasma membrane of BEC lining bile ducts, but was more diffusely expressed throughout the cytoplasm of cholangioles. In conclusion, BEC IL-6/gp130/STAT3 signaling noncoordinately upregulates BEC SPRR2 that appears to contribute to modification of the biliary barrier under conditions of stress.