Anna Makowska

Mesenchymal Stem Cells from Rats with Chronic Kidney Disease Exhibit Premature Senescence and Loss of Regenerative Potential

Mesenchymal stem cell (MSC) transplantation has the potential for organ repair. Nevertheless, some factors might lessen the regenerative potential of MSCs, e.g. donor age or systemic disease. It is thus important to carefully assess the patients suitability for autologous MSC transplantation. Here we investigated the effects of chronic kidney disease (CKD) on MSC function. We isolated bone marrow MSCs from remnant kidney rats (RK) with CKD (CKD-RK-MSC) and found signs of premature senescence: spontaneous adipogenesis, reduced proliferation capacity, active senescence-associated-β-galactosidase, accumulation of actin and a modulated secretion profile. The functionality of CKD-RK-MSCs in vivo was tested in rats with acute anti-Thy1.1-nephritis, where healthy MSCs have been shown to be beneficial. Rats received healthy MSCs, CKD-RK-MSC or medium by injection into the left renal artery. Kidneys receiving healthy MSCs exhibited accelerated healing of glomerular lesions, whereas CKD-RK-MSC or medium exerted no benefit. The negative influence of advanced CKD/uremia on MSCs was confirmed in a second model of CKD, adenine nephropathy (AD). MSCs from rats with adenine nephropathy (CKD-AD-MSC) also exhibited cellular modifications and functional deficits in vivo. We conclude that CKD leads to a sustained loss of in vitro and in vivo functionality in MSCs, possibly due to premature cellular senescence. Considering autologous MSC therapy in human renal disease, studies identifying uremia-associated mechanisms that account for altered MSC function are urgently needed.

Long‐term potentiation of orofacial sensorimotor processing by noxious input from the semispinal neck muscle in mice

Tension-type headache is the most common type of primary headaches but no conclusive concept of pathophysiology exists. This may be due to a lack of an appropriate animal model. This study addressed the hypothesis that noxious neck muscle input induces central sensitization of orofacial sensorimotor processing. The effect of hypertonic saline injection into the semispinal neck muscle on the jaw-opening reflex (JOR) was investigated in anaesthetized mice (n = 11). Hypertonic saline injection into the neck muscle facilitated the JOR for at least one hour: integral (+94.5%) and duration (+18.7%) increased, latency decreased (-7.5%). The reflex threshold decreased to 61% after injection. Isotonic saline injection into the neck muscle (n = 11) or hypertonic saline injection into a hindpaw muscle (n = 10) did neither change the reflex integral nor the threshold. Long-term potentiation of the JOR by noxious neck muscle input may be an appropriate model to investigate tension-type headache pathophysiology.

ATP induces sustained facilitation of craniofacial nociception through P2X receptors on neck muscle nociceptors in mice

Noxious input from neck muscles probably plays a key role in tension-type headache pathophysiology. ATP selectively excites group III and IV muscle afferents in vitro. Accordingly, ATP infusion into trapezius muscle induces strong pain and local tenderness in healthy man. The present study addresses the impact of ATP on neck muscle nociception in anaesthetized mice. Craniofacial nociceptive processing was tested by the jaw-opening reflex via noxious electrical tongue stimulation. Within 2 h after injection of 100 nmol/l or 1 μmol/l ATP into semispinal neck muscles, reflex integrals significantly increased by 114% or 328%, respectively. Preceding intramuscular administration of the P2X receptor antagonist PPADS (3–100 nmol/l) suppressed the ATP effect. Subsequent application of PPADS (100 nmol/l) caused a total recovery of facilitated reflex to baseline values. ATP induces sustained facilitation of craniofacial nociception by prolonged excitation of P2X receptors in neck muscles.

Nerve growth factor injection into semispinal neck muscle evokes sustained facilitation of the jaw-opening reflex in anesthetized mice -- possible implications for tension-type headache.

Nociceptive input from neck muscles probably plays a role in the pathophysiology of tension-type headache. In order to elaborate an animal model, the impact of noxious input from neck muscles on orofacial sensorimotor processing was investigated by electrophysiological means in anesthetized mice. Group IV muscle afferents of the semispinal neck muscle were excited by local injection of nerve growth factor (NGF, 0.8 microM, 20 microl). Orofacial sensorimotor processing was monitored by the jaw-opening reflex (JOR) elicited by electric tongue stimulation. After unilateral NGF injection into the right neck muscle (n = 10), JOR integral (+89%) and duration (+9%) increased and latency decreased (-5%) for at least 1 h. Bilateral injection of NGF (n = 10) into neck muscles induced an increase of JOR integral (+111%) and duration (+20%) and a reduction of latency (-9%). This facilitation of the JOR lasted for at least 90 min without any downward drift (n = 5). Electric JOR threshold diminished after NGF injection. After intramuscular injection of isotonic saline into the right semispinal neck muscle (20 microl), the JOR remained unchanged (n = 10). Local NGF injection into neck muscles evoked noxious input to the brainstem that induced a sustained central facilitation of the JOR for more than 1 h. This long-term facilitation of orofacial sensorimotor processing by a singular NGF injection possibly reflects plastic changes of nociceptive synaptic processing that may be involved in the pathophysiology of headache.

Nerve growth factor and ATP excite different neck muscle nociceptors in anaesthetized mice

Neck muscle nociception probably plays a major role in the pathophysiology of tension-type headache. Recent studies have demonstrated sustained facilitation of brainstem nociception due to noxious neck muscle input evoked by nerve growth factor (NGF) or α,β-methylene ATP (ATP) in mice. Hypothesized different afferent pathways in NGF and ATP models were addressed by local application of tetrodotoxin (TTX) in neck muscles. Brainstem nociception was monitored in 55 anaesthetized mice by the jaw-opening reflex elicited by electrical tongue stimulation. Sole administration of 100nmol/l ATP or 0.8 μmol/l NGF evoked sustained reflex facilitation for at least 95 min. Preceding TTX administration prevented ATP-induced facilitation, but was without effect on NGF. Subsequent administration of 100 nmol/l TTX reversed ATP-evoked facilitation, but was ineffective on NGF. Divergent effects of TTX suggest preferential excitation of group III muscle afferents by ATP and group IV by NGF. Thus, both models address different pathways in pericranial pain.

IL6 secreted by Ewing sarcoma tumor microenvironment confers anti-apoptotic and cell-disseminating paracrine responses in Ewing sarcoma cells

BackgroundThe prognosis of patients with Ewing sarcoma (ES) has improved over the course of the last decades. However, those patients suffering from metastatic and recurrent ES still have only poor chances of survival and require new therapeutic approaches. Interleukin-6 (IL6) is a pleiotropic cytokine expressed by immune cells and a great variety of cancer cells. It induces inflammatory responses, enhances proliferation and inhibits apoptosis in cancer cells, thereby promoting chemoresistance.MethodsWe investigated expression of IL6, its receptors and the IL6 signal transduction pathway in ES tumor samples and cell lines applying reverse transcriptase PCR, immunoblot and immunohistochemistry. The impact of IL6 on cell viability and apoptosis in ES cell lines was analyzed by MTT and propidium iodide staining, migration assessed by chorioallantoic membrane (CAM) assay.ResultsImmunohistochemistry proved IL6 expression in the stroma of ES tumor samples. IL6 receptor subunits IL6R and IL6ST were expressed on the surface of ES cells. Treatment of ES cells with rhIL6 resulted in phosphorylation of STAT3. rhIL6 protected ES cells from serum starvation-induced apoptosis and promoted migration. IL6 blood serum levels were elevated in a subgroup of ES patients with poor prognosis.ConclusionsThese data suggest that IL6 contributes to ES tumor progression by increasing resistance to apoptosis in conditions of cellular stress, such as serum starvation, and by promotion of metastasis.

Inhibition of nitric oxide synthases prevents and reverses α,β-meATP-induced neck muscle nociception in mice:

Introduction: Tension-type headache (TTH) is associated with noxious input from neck muscles. Intravenous administration of the unspecific nitric oxide synthase inhibitor L-NMMA in chronic TTH patients caused analgesia and reduction of neck muscle tenderness. Methods: The unspecific nitric oxide synthase inhibitor L-NMMA was applied in an experimental model for neck muscle nociception in anesthetized mice (N = 25). Results: Local injection of α,β-meATP into semispinal neck muscles induced sustained facilitation of brainstem nociception as monitored by the jaw-opening reflex. Preceding intraperitoneal administration of L-NMMA (0.05, 0.1, 1 mg/kg) prevented reflex facilitation evoked by α,β-meATP in a dose-dependent manner. Intraperitoneal injection of L-NMMA subsequent to intramuscular α,β-meATP application reversed established brainstem reflex facilitation back to baseline values. Discussion: Both experiments with preceding and subsequent L-NMMA indicate the involvement of nitric oxide synthases in the induction and maintenance of facilitation. However, future experiments will have to address the involvement of various isoenzymes in order to provide for new therapeutic concepts in TTH.

Chloroquine Sensitizes Nasopharyngeal Carcinoma Cells but Not Nasoepithelial Cells to Irradiation by Blocking Autophagy

Background Treatment of nasopharyngeal carcinoma requires the application of high dosages of radiation, leading to severe long-term complications in the majority of patients. Sensitizing tumor cells to radiation could be a means to increase the therapeutic window of radiation. Nasopharyngeal carcinoma cells display alterations in autophagy and blockade of autophagy has been shown to sensitize them against chemotherapy. Methods We investigated the effect of chloroquine, a known inhibitor of autophagy, on sensitization against radiation-induced apoptosis in a panel of five nasopharyngeal carcinoma cell lines and a SV40-transformed nasoepithelial cell line. Autophagy was measured by immunoblot of autophagy-related proteins, immunofluorescence of autophagosomic microvesicles and electron microscopy. Autophagy was blocked by siRNA against autophagy-related proteins 3, 5, 6 and 7 (ATG3, ATG5, ATG6 and ATG7). Results Chloroquine sensitized four out of five nasopharyngeal cancer cell lines towards radiation-induced apoptosis. The sensitizing effect was based on the blockade of autophagy as inhibition of ATG3, ATG5, ATG6 and ATG7 by specific siRNA could substitute for the effect of chloroquine. No sensitization was seen in nasoepithelial cells. Conclusion Chloroquine sensitizes nasopharyngeal carcinoma cells but not nasoepithelial cells towards radiation-induced apoptosis by blocking autophagy. Further studies in a mouse-xenograft model are warranted to substantiate this effect in vivo.

Interferon beta induces apoptosis in nasopharyngeal carcinoma cells via the TRAIL-signaling pathway

The combination of neoadjuvant chemotherapy, radiochemotherapy, and maintenance therapy with interferon beta (IFNβ) has led to superior results in the treatment of children and adolescents with nasopharyngeal carcinoma (NPC). However, nothing is known about the mechanism of the antitumor activity of IFNβ in NPC. Here, we investigate the role of IFNβ on apoptosis in NPC cells. Six NPC cell lines, one patient-derived NPC xenograft (PDX) and one SV40-transformed nasoepithelial cell line were used. Induction of apoptosis by IFNβ was measured by flow cytometric analysis of subG1-DNA-content, Hoechst 33258 staining and activation of caspase-3. Dissection of death ligand signaling pathways included measuring surface expression of its components by flow cytometry, activation by death ligands and neutralization with specific antibodies and siRNA. IFNβ induced apoptosis at concentrations achievable in humans in five of six NPC cell lines and in PDX cells but not in nasoepithelial cells. Inhibition of caspases-3 and −8 abrogated this effect suggesting IFNβ promoted apoptosis through the extrinsic pathway. IFNβ induced surface expression of TRAIL and TRAIL-R2 and the addition of an anti-TRAIL-antibody or transfection with TRAIL-siRNA blocked IFNβ-induced apoptosis. No induction of TRAIL-expression was noted in the IFNβ-resistant cell line. In conclusion, IFNβ leads to apoptosis in NPC cells in an autocrine way via the induction of TRAIL expression and subsequent activation of the TRAIL-signaling pathway. The mechanism described could at least partly explain the clinical benefit of IFNβ in the treatment of NPC. Further studies in a mouse-xenograft model are warranted to substantiate this effect in vivo.