Chronic Inflammation and Angiogenic Signaling Axis Impairs Differentiation of Dental-Pulp Stem Cells

<div><p>Dental-pulp tissue is often exposed to inflammatory injury. Sequested growth factors or angiogenic signaling proteins that are released following inflammatory injury play a pivotal role in the formation of reparative dentin. While limited or moderate angiogenesis may be helpful for dental pulp maintenance, the induction of significant level of angiogenesis is probably highly detrimental. Hitherto, several studies have addressed the effects of proinflammatory stimuli on the survival and differentiation of dental-pulp stem cells (DPSC), <i>in vitro</i>. However, the mechanisms communal to the inflammatory and angiogenic signaling involved in DPSC survival and differentiation remain unknown. Our studies observed that short-term exposure to TNF-α (6 and 12 hours [hrs]) induced apoptosis with an upregulation of VEGF expression and NF-κB signaling. However, long-term (chronic) exposure (14 days) to TNF-α resulted in an increased proliferation with a concomitant shortening of the telomere length. Interestingly, DPSC pretreated with Nemo binding domain (NBD) peptide (a cell permeable NF-κB inhibitor) significantly ameliorated TNF-α- and/or VEGF-induced proliferation and the shortening of telomere length. NBD peptide pretreatment significantly improved TNF-α-induced downregulation of proteins essential for differentiation, such as bone morphogenic proteins (BMP)-1 & 2, BMP receptor isoforms-1&2, trasnforming growth factor (TGF), osteoactivin and osteocalcin. Additionally, inhibition of NF-κB signaling markedly increased the mineralization potential, a process abrogated by chronic exposure to TNF-α. Thus, our studies demonstrated that chronic inflammation mediates telomere shortening via NF-κB signaling in human DPSC. Resultant chromosomal instability leads to an emergence of increased proliferation of DPSC, while negatively regulating the differentiation of DPSC, <i>in vitro</i>.</p></div

The Human Primer Sequences used for Real-Time PCR.

<p>The Human Primer Sequences used for Real-Time PCR.</p

Inhibition of NF-κB signaling impedes TNF-α-induced increase in proliferation and angiogenic signaling.

<p>(<b>A</b>) Histogram plots from flow cytometry analysis show the percentage of Live/Dead (L/D) cells in the presence of vehicle control or NBD peptide. (<b>B</b>) MTT assay showing a decrease in TNF-α-induced increase in proliferation, in DPSC challenged with varying concentrations of NBD domain peptide (5 and 10 µM). (<b>C</b>) Real time PCR analysis showing the RNA expression levels of VEGF, EGF, FGF-1, and FGF-2 in DPSC treated with TNF-α in the absence or presence of NBD (5 and 10 µM). DPSC treated with NBD alone serve as a positive control. (<b>D</b>) Average telomere length was measured from total genomic DNA by using a sequence-independent multiplex qPCR technique. Data shown are the mean telomere length quantified, at day 10, in DPSC treated with TNF-α in the absence or presence of NBD. The data shown are Mean ± SD. *p<0.05 from three independent experiments. “NS” represents a non-significant difference between the test groups.</p

Short-term exposure to TNF-α induces apoptosis via NF-κB signaling with an associated increase in VEGF expression in DPSC.

<p>(<b>A</b>) DPSC were cultured in 3% serum containing medium for 0, 4, and 6 hrs in the absence or presence of TNF-α, and cell viability was assessed using MTT assay. (<b>B</b>) DPSC grown to approximately 80% confluence was immunostained for propidium iodide in the absence or presence of TNF-α at 0, 4, and 6 hrs. (<b>C</b>) Representative Western blot data showing an increase in Caspase 3 expression upon challenging with varying concentrations of TNF-α (0, 5, and 10 ng/ml) at 4 and 6 hrs. (<b>D</b>) Western blot analysis of extracts from DPSC showing an increase in the expression of phospho-p65 (upper panel) upon challenging with TNF-α for varying time points. (<b>E</b>) Western blot analysis showing the expression levels of phospho-I-κB-α and I-κB-α. (<b>F</b>) VEGF and FGF levels in DPSC challenged with TNF-α for 0, 4, 6, and 12 hrs in 3% serum containing medium. Note an increase in the expression of VEGF and FGF following TNF-α treatment at 6 and 12 hrs. The data shown are Mean ± SD. *p<0.05.</p

Pathogenic Role of Store-Operated and Receptor-Operated Ca2+ Channels in Pulmonary Arterial Hypertension

Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness are the major causes for the elevated pulmonary vascular resistance (PVR) in patients with PAH. The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca2+ signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca2+]cyt in PASMC is increased by Ca2+ influx through Ca2+ channels in the plasma membrane and by Ca2+ release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca2+ entry pathways, voltage-dependent Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) and voltage-independent Ca2+ influx through store-operated Ca2+ channels (SOC) and receptor-operated Ca2+ channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH

Signaling pathway downstream of inflammatory mediator (TNF-α) signaling in human DPSC.

<p>Prolonged exposure to TNF-α induce NF-κB signaling axis, which in turn induce an enhanced antiapoptotic gene expression and decreased telomere length. The resultant chromosomal instability leads to an increase in the proliferation of DPSC, with an impaired differentiation potential.</p

NF-κB inhibition ameliorates TNF-α-induced changes in mineralization-associated proteins and mineralized nodule formation.

<p>(<b>A</b>) Mineralized nodule formation was assessed by ALR staining. DPSC challenged with TNF-α and VEGF in the absence or presence of NBD was visualized for ALR staining at day 14. NBD was replaced every 4 days (at least 3 times during the mineralization process). (<b>B</b>) Alkaline phosphatase enzyme activity was quantified in DPSC treated with TNF-α and VEGF, in the absence or presence of NBD. (<b>C</b>) Real time PCR analysis showing the expression levels of the differentiation markers; osteocalcin, osteoactivin, and Runx2 in cells challenged with TNF-α and VEGF, in the absence or presence of NBD. The data shown are Mean ± SD. *p<0.05, from at least three independent experiments. “NS” represents a non-significant difference between the test groups.</p

Targeted Gene Inactivation of Calpain-1 Suppresses Cortical Degeneration Due to Traumatic Brain Injury and Neuronal Apoptosis Induced by Oxidative Stress*

Calpains are calcium-regulated cysteine proteases that have been implicated in the regulation of cell death pathways. Here, we used our calpain-1 null mouse model to evaluate the function of calpain-1 in neural degeneration following a rodent model of traumatic brain injury. In vivo, calpain-1 null mice show significantly less neural degeneration and apoptosis and a smaller contusion 3 days post-injury than wild type littermates. Protection from traumatic brain injury corroborated with the resistance of calpain-1 neurons to apoptosis induced by oxidative stress. Biochemical analysis revealed that caspase-3 activation, extracellular calcium entry, mitochondrial membrane permeability, and release of apoptosis-inducing factor from mitochondria are partially blocked in the calpain-1 null neurons. These findings suggest that the calpain-1 knock-out mice may serve as a useful model system for neuronal protection and apoptosis in traumatic brain injury and other neurodegenerative disorders in which oxidative stress plays a role