Hitendra S. Chand

Structure, function and biology of tissue factor pathway inhibitor-2

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region,three tandem Kunitz-type domains and a positively charged carboxy-terminal tail. Human TFPI-2, previously designated as placental protein 5, inhibits a broad spectrum of serine proteinases almost exclusively through its first Kunitz-type domain, and is thought to play an important role in the regulation of extracellular matrix digestion and remodeling. In this context, reduced synthesis of TFPI-2 has been related to numerous pathophysiological processes such as inflammation, angiogenesis, atherosclerosis, retinal degeneration and tumor growth/metastasis. In this review, we document current information regarding the expression of TFPI-2 by various tissues, its inhibitory activity and proteinase specificity in-vitro, and discuss possible physiological roles for this inhibitor based on in-vivo studies.

Identification of a novel human tissue factor splice variant that is upregulated in tumor cells

Tissue factor (TF) is a transmembrane glycoprotein that serves as the prime initiator of blood coagulation and plays a critical role in thrombosis and hemostasis. In addition, a variety of tumor cells overexpress cell‐surface TF, which appears to be important for tumor angiogenesis and metastasis. To elucidate the mechanism involved in the upregulation of TF in human tumor cells, a comprehensive analysis of TF mRNA from various normal and tumor cells was performed. The results of these studies indicate that, in addition to possessing a normal full‐length TF transcript and minor levels of an alternatively spliced transcript known as alternatively‐spliced tissue factor (asTF) (Bogdanov et al., Nat Med 2003;9:458–62), human tumor cells express additional full‐length TF transcripts that are also generated by alternative splicing. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) and 5′‐rapid amplification of cDNA ends‐ (5′‐RACE) based analyses of cytoplasmic RNA from normal and tumor cells revealed that there is alternative splicing of the first intron between exon I and exon II resulting in 2 additional TF transcripts. One of the transcripts has an extended exon I with inclusion of most of the first TF intron (955 bp), while the second transcript is formed by the insertion of a 495 bp sequence, referred to as exon IA, derived from an internal sequence of the first intron. The full length TF transcript with alternatively spliced novel exon IA, referred to as alternative exon 1A‐tissue factor (TF‐A), represented ˜1% of the total TF transcripts in normal cells, but constituted 7–10% of the total TF transcript in tumor cells. Quantitative real‐time RT‐PCR analysis indicated that cultured human tumor cells contain 10–25‐fold more copy numbers of TF‐A in comparison to normal, untransformed cells. We propose that high‐level expression of the novel TF‐A transcript, preferentially in tumor cells, may have utility in the diagnosis and staging of a variety of solid tumors.

Molecular Processes that Drive Cigarette Smoke–Induced Epithelial Cell Fate of the Lung

Cigarette smoke contains numerous chemical compounds, including abundant reactive oxygen/nitrogen species and aldehydes, and many other carcinogens. Long-term cigarette smoking significantly increases the risk of various lung diseases, including chronic obstructive pulmonary disease and lung cancer, and contributes to premature death. Many in vitro and in vivo studies have elucidated mechanisms involved in cigarette smoke-induced inflammation, DNA damage, and autophagy, and the subsequent cell fates, including cell death, cellular senescence, and transformation. In this Translational Review, we summarize the known pathways underlying these processes in airway epithelial cells to help reveal future challenges and describe possible directions of research that could lead to better management and treatment of these diseases.

Discriminating Virulence Mechanisms among Bacillus anthracis Strains by Using a Murine Subcutaneous Infection Model

ABSTRACT Bacillus anthracis strains harboring virulence plasmid pXO1 that encodes the toxin protein protective antigen (PA), lethal factor, and edema factor and virulence plasmid pXO2 that encodes capsule biosynthetic enzymes exhibit different levels of virulence in certain animal models. In the murine model of pulmonary infection, B. anthracis virulence was capsule dependent but toxin independent. We examined the role of toxins in subcutaneous (s.c.) infections using two different genetically complete (pXO1+ pXO2+) strains of B. anthracis, strains Ames and UT500. Similar to findings for the pulmonary model, toxin was not required for infection by the Ames strain, because the 50% lethal dose (LD50) of a PA-deficient (PA−) Ames mutant was identical to that of the parent Ames strain. However, PA was required for efficient s.c. infection by the UT500 strain, because the s.c. LD50 of a UT500 PA− mutant was 10,000-fold higher than the LD50 of the parent UT500 strain. This difference between the Ames strain and the UT500 strain could not be attributed to differences in spore coat properties or the rate of germination, because s.c. inoculation with the capsulated bacillus forms also required toxin synthesis by the UT500 strain to cause lethal infection. The toxin-dependent phenotype of the UT500 strain was host phagocyte dependent, because eliminating Gr-1+ phagocytes restored virulence to the UT500 PA− mutant. These experiments demonstrate that the dominant virulence factors used to establish infection by B. anthracis depend on the route of inoculation and the bacterial strain.

Deacetylation of p53 induces autophagy by suppressing Bmf expression

IFN-γ induces the interaction of HDAC1 and p53, leading to p53 deacetylation, which facilitates autophagy via Bmf suppression.

Acute Inflammation Induces Insulin-like Growth Factor-1 to Mediate Bcl-2 and Muc5ac Expression in Airway Epithelial Cells

Generally, exposure to LPS in human airways occurs in the form of aerosols and causes an acute inflammatory response or exacerbates existing chronic inflammatory conditions by enhancing airway remodeling and associated pathologies. The present study evaluated which inflammatory mediators may be responsible for the expression of Bcl-2 and mucus cell metaplasia when mice are exposed to aerosolized LPS. At 3 days after exposure, aerosolized LPS (for 20-40 min) with the estimated lung deposited dosage of 0, 0.02, 0.2, 1.4, and 20.2 μg showed a characteristic dose-dependent increase in polymorphonuclear neutrophils. Significant increases of proinflammatory mediators, including IL-1β, TNF-α, IL-6, growth-related oncogene or keratinocyte-derived cytokine, IFN-γ-induced protein-10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1α, were detected at the highest doses. In addition to increased numbers of airway epithelial cells, mucus cell numbers and mucus production were increased in a dose-dependent manner. Hyperplastic epithelial cells expressed insulin-like growth factor (IGF)-1 and, similar to previous studies, increased expression of the prosurvival protein Bcl-2 and induced expression of Muc5ac. Suppression of IGF-1 expression using retroviral shRNA blocked Bcl-2 expression in human and murine airway epithelial cells and Muc5ac in primary murine airway epithelial cells. These findings show that acute inflammation induces IGF-1 to mediate Bcl-2 and Muc5ac expression in airway epithelial cells.

Intracellular Insulin-like Growth Factor-1 Induces Bcl-2 Expression in Airway Epithelial Cells

Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and it can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and insulin-like growth factor-1 (IGF-1) coincided with induced Bcl-2 expression compared with controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using short hairpin RNA showed that intracellular IGF-1 (IC-IGF-1) was increasing Bcl-2 expression. Blocking epidermal growth factor receptor or IGF-1R activation also suppressed IC-IGF-1 and abolished the Bcl-2 induction. Induced expression and colocalization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and epidermal growth factor receptor pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases.

Anti-IgE therapy results in decreased myeloid dendritic cells in asthmatic airways

Anti-IgE treatment of intermittent to mild-persistent asthma in a cohort of seven volunteers resulted in improved allergen-induced airway hyperreactivity and a significant reduction in the number of airway myeloid dendritic cells.

Human tissue factor pathway inhibitor-2 does not bind or inhibit activated matrix metalloproteinase-1

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor associated with the extracellular matrices of vascular cells. A recent report provided in vitro evidence that TFPI-2 may be a novel inhibitor of the matrix metalloproteinases MMP-1, MMP-13, MMP-2 and MMP-9. In studies aimed at identifying the structural elements of TFPI-2 mediating the putative inhibition of the above MMPs, we re-examined the ability of native TFPI-2 to form complexes with MMP-2, MMP-9 and MMP-1, as well as assess its ability to inhibit the proteolytic activity of the interstitial collagenase, activated MMP-1. We report here that TFPI-2 failed to form complexes with MMP-2, MMP-9 and MMP-1 as revealed in immunoprecipitation and ligand blotting studies. In addition, TFPI-2 had no influence on the proteolytic activity of activated MMP-1 towards triple-helical collagen. These data provide presumptive evidence that TFPI-2 does not bind to MMP-2, MMP-9 and MMP-1, or regulate MMP-1, in the extracellular matrix.

Gestational exposure of mice to secondhand cigarette smoke causes bronchopulmonary dysplasia blocked by the nicotinic receptor antagonist mecamylamine.

Background: Cigarette smoke (CS) exposure during gestation may increase the risk of bronchopulmonary dysplasia (BPD)—a developmental lung condition primarily seen in neonates that is characterized by hypoalveolarization, decreased angiogenesis, and diminished surfactant protein production and may increase the risk of chronic obstructive pulmonary disease. Objective: We investigated whether gestational exposure to secondhand CS (SS) induced BPD and sought to ascertain the role of nicotinic acetylcholine receptors (nAChRs) in this response. Methods: We exposed BALB/c and C57BL/6 mice to filtered air (control) or SS throughout the gestation period or postnatally up to 10 weeks. Lungs were examined at 7 days, 10 weeks, and 8 months after birth. Results: Gestational but not postnatal exposure to SS caused a typical BPD-like condition: suppressed angiogenesis [decreased vascular endothelial growth factor (VEGF), VEGF receptor, and CD34/CD31 (hematopoietic progenitor cell marker/endothelial cell marker)], irreversible hypoalveolarization, and significantly decreased levels of Clara cells, Clara cell secretory protein, and surfactant proteins B and C, without affecting airway ciliated cells. Importantly, concomitant exposure to SS and the nAChR antagonist mecamylamine during gestation blocked the development of BPD. Conclusions: Gestational exposure to SS irreversibly disrupts lung development leading to a BPD-like condition with hypoalveolarization, decreased angiogenesis, and diminished lung secretory function. Nicotinic receptors are critical in the induction of gestational SS–induced BPD, and the use of nAChR antagonists during pregnancy may block CS-induced BPD.