laser-doppler-tool-for-measuring-pancreatic-islet-microvascular

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion <em>In Vivo</em>

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

Chinese Academy of Medical Sciences & Peking Union Medical College

This paper aimed at investigating the properties of cultured cardiomyocytes using microcirculatory and molecular technology to culture cardiomyocytes from different parts of the neonate Wistar rat's heart and record their spontaneous pulsation under time-lapse video microscopy, then analyze their activity and inspect their survival rate and apoptotic rate under natural and nitric oxide conditions. The pulsation frequency in cardiomtocytes of different parts in heart are: 78.5+/-11.0 beats/min in the atrium, 88.4+/-6.3 beats/min in the left ventricle, 90.3+/-7.9 beats/min in the right ventricle and 115.3 +/-11.4 beats/min in the cardiac apex, respectively, with an average frequency of 81.3 beats/min. Different concentrations of nitric oxide showed no effect on the frequency of cardiomyocyte pulsation. The survival rates of the above cardiomyocytes are 96.0%, 95.0%, 95.0%, and 95.3% respectively and 95.0% for the whole heart. The apoptotic rates are 1.3%, 1.1%, 4.8%, and 1.8% respectively and 5.1% for the whole heart. Different concentrations of nitric oxide had no effect on these results. Our study showed that cultured myocardial cells from different parts of the heart displayed various pulsation frequencies, and the frequency of the cardiac apex is the highest while the atrium is lowest. We also found that there is no statistically significant difference in the survival rates and apoptotic rates of different parts of the heart, and that nitric oxide has no effect on the beating frequency, survival rates or apoptotic rates of the cardiomyocytes in vitro.

Experimental study on the function of cardiomyocytes.

This study was designed to observe the effect of tumor conditioned medium (TCM) on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then we measured their proliferation, apoptosis and cell cycle distribution by MTT and flow cytometery (FCM). Following the stimulation of TCM, HUVECs showed higher pro-mitogenic and anti-apoptotic ability than did the negative control group (ECGF-free medium with 20% FBS), but a similar ability to the positive control group (medium with ECGF and 20% FBS). From these results, we can conclude that breast carcinoma cell line MDA-MB-231 could secret soluble pro-angiogenic factors that induce HUVEC angiogenic switching, including cell cycle progression, proliferation and growth. The role and character of these factors remain to be further studied.

The mitogenic and anti-apoptotic activity of tumor conditioned medium on endothelium.

This study was designed to comprehensively analyze the differential expression of proteins from human umbilical vein endothelial cells (HUVECs) exposed to tumor conditioned medium (TCM) and to identify the key regulator in the cell cycle progression. The HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then their cell cycle distribution was measured by flow cytometer (FCM). The role of protein in cell cycle progression was detected via two-dimensional polyacrylamide gel electrophoresis (2-DE) and western blotting. Following the stimulation of TCM, HUVECs showed a more cells in the S phase than did the negative control group (ECGF-free medium with 20% FBS), but the HUVECs' level was similar to the positive control group (medium with 25 micrograms/ml ECGF and 20% FBS). Increased expression of cyclin D1/E and some changes in other related proteins occurred after incubation with TCM. From our results, we can conclude that breast carcinoma cell line MDA-MB-231 may secrete soluble pro-angiogenic factors that induce the HUVEC angiogenic switch, during which the expression of cell cycle regulator cyclin D1/E increases and related proteins play an important role in this process.

A proteomic study on cell cycle progression of endothelium exposed to tumor conditioned medium and the possible role of cyclin D1/E.

Melatonin is the major secretory product of the pineal gland and is considered an important natural oncostatic agent. The anticancer activity of melatonin is due to its immunomodulatory, anti-proliferative and antioxidative effects. At present there are no direct data available as to melatonin's possible influence on angiogenesis, which is a major biological mechanism responsible for tumor growth and dissemination. The current study investigated the influence of melatonin on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were cultured, identified, and purified. Cell growth and viability, DNA fragmentation and cell cycle analyses were determined. To elucidate the mechanism of action of melatonin, Western blot analyses for P53, Bax and Bcl-2 expression were carried out. The results demonstrate the anti-proliferative and apoptosis-inducing effects of melatonin; these changes were associated with cell cycle arrest, upregulation of P53 and Bax and downregulation of Bcl-2. Taken together, our data showed that melatonin in high concentrations markedly reduces HUVECs proliferation, induces cellular apoptosis, and modulates cell cycle length. P53 and Bax/Bcl-2 expression changes may be involved in these actions of melatonin.

Effect and mechanism of melatonin's action on the proliferation of human umbilical vein endothelial cells.

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma) such as prostaglandin metabolite 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) or thiazolidinedione pioglitazone have been identified as a new class of anti-inflammatory compounds with possible clinical applications. Reactive oxygen species play an important role in the generation of cellular damage by induction of proinflammatory cytokines and chemokines during myocardial I/R. These events were preceded by activation of the transcription factors nuclear factor (NF)-kappaB pathway. It has been suggested that myocardium overproduces TNF-alpha after I/R, and locally produced TNF-alpha is sufficient to cause severe impairment of cardiac function. LPS-induced CXC chemokine (LIX) is a rodent chemokine with potent neutrophil-chemotactic activity. Based on this concept, we examined the effects of 15d-PGJ2 and pioglitazone on oxidative stress-induced TNF-alpha and LIX expression in neonatal rat cardiac myocytes. Pretreatment of myocytes with 15d-PGJ2 or pioglitazone decreased hydrogen peroxide-induced TNF-alpha and LIX production (mRNA and protein) in a concentration-dependent manner. The beneficial effects of both ligands were associated with reduction of hydrogen peroxide-induced NF-kappaB activation. Treatment with 15d-PGJ2, but not pioglitazone, caused dose-dependent activation of heat shock factor 1, which could render cells unresponsive to stimulation of NF-kappaB. The cytoprotection afforded by pioglitazone was attenuated by the PPAR-gamma antagonist GW9662, which failed to affect the beneficial effects afforded by 15d-PGJ2. Taken together, these results demonstrate that treatment with these chemically distinct ligands of PPAR-gamma results in diverse anti-inflammatory mechanisms.

Peroxisome proliferator-activated receptor-gamma ligands 15-deoxy-delta(12,14)-prostaglandin J2 and pioglitazone inhibit hydroxyl peroxide-induced TNF-alpha and lipopolysaccharide-induced CXC chemokine expression in neonatal rat cardiac myocytes.

Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP) cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2-4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC) drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assay(EMSA), we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia.

SALL4, a stem cell factor, affects the side population by regulation of the ATP-binding cassette drug transport genes.

Aggressive cancers and embryonic stem (ES) cells share a common gene expression signature. Identifying the key factors/pathway(s) within this ES signature responsible for the aggressiveness of cancers can lead to a potential cure. In this study, we find that SALL4, a gene involved in the maintenance of ES cell self-renewal, is aberrantly expressed in 47.7% of primary human endometrial cancer samples. It is not expressed in normal or hyperplastic endometrial. More importantly, SALL4 expression is positively correlated with worse patient survival and aggressive features such as metastasis in endometrial carcinoma. Further functional studies have shown that loss of SALL4 inhibits endometrial cancer cell growth in vitro and tumorigenicity in vivo, as a result of inhibition of cell proliferation and increased apoptosis. In addition, downregulation of SALL4 significantly impedes the migration and invasion properties of endometrial cancer cells in vitro and their metastatic potential in vivo. Furthermore, manipulation of SALL4 expression can affect drug sensitivity of endometrial cancer cells to carboplatin. Moreover, we show that SALL4 specifically binds to the c-Myc promoter region in endometrial cancer cells. While downregulation of SALL4 leads to a decreased expression of c-Myc at both protein and mRNA levels, ectopic SALL4 overexpression causes increased c-Myc protein and mRNA expression, indicating that c-Myc is one of the SALL4 downstream targets in endometrial tumorigenesis. In summary, we are the first to demonstrate that SALL4 has functional role(s) in metastasis and drug resistance in aggressive endometrial cancer. As a consequence of its functional roles in cancer cell and absence in normal tissue, SALL4 is a potential novel therapeutic target for the high-risk endometrial cancer patient population.

SALL4 is a new target in endometrial cancer.

The microcirculation plays an important role in the pathogenesis of diabetes and its complications. We hypothesized that pancreatic islet microvascular (PIM) vasomotion, as a parameter of pancreatic islet microcirculation function, is abnormal in diabetic mice and that insulin treatment may reverse this dysfunction.

Insulin treatment restores islet microvascular vasomotion function in diabetic mice.

BST-2 (tetherin, CD317, and HM1.24) is induced by interferon and restricts virus release by tethering the enveloped viruses to the cell surface. The effect of BST-2 on influenza A virus (IAV) infection has been inconclusive. In the present study, we report that BST-2 diminishes the production of IAV virus-like particles (VLPs) that are generated by viral neuraminidase and hemagglutinin proteins to a much greater degree than it inhibits the production of wild-type IAV particles. This relatively weaker inhibition of IAV is associated with reduction in BST-2 levels, which is caused by the M2 protein that interacts with BST-2 and leads to down-regulation of cell surface BST-2 via the proteasomal pathway. Similarly to the viral antagonist Vpu, M2 also rescues the production of human immunodeficiency virus-1 VLPs and IAV VLPs in the presence of BST-2. Replication of wild-type and the M2-deleted viruses were both inhibited by BST-2, with the M2-deleted IAV being more restricted. These data reveal one mechanism that IAV employs to counter restriction by BST-2.

BST-2 restricts IAV release and is countered by the viral M2 protein.

Accumulating evidence demonstrates that microcirculation plays a role in the pathogenesis of hypertension. In the current study, we demonstrated that pancreatic islet microvascular vasomotion of spontaneously hypertensive rats (SHRs) lost the ability to regulate blood flow perfusion and exhibited a lower microvascular blood perfusion pattern which was negative correlated with blood glucose level. SHRs administrated with insulin revealed an improvement of pancreatic islet microvascular vasomotion and blood perfusion pattern. In vitro, the expressions of endothelial nitric oxide synthase (eNOS) and phospho-eNOS(p-eNOS) were significantly decreased in high glucose exposed islet endothelial cells (iECs), accompanied with a higher ratio of eNOS monomer to eNOS dimer and a significantly increased malondialdehyde and nitrite levels. Meanwhile, barrier function, tube formation and migration capacities of high glucose exposed iECs were significantly inhibited. In contrast, iECs dysfunction induced by glucose toxicity and oxidative stress was attenuated or improved by supplement with insulin, L-arginine and β-mercaptoethanol. In summary, our findings suggest that functional status of pancreatic islet microvascular vasomotion is impaired in SHRs and provide evidence that treatment with insulin, L-arginine and β-mercaptoethanol improves endothelium-dependent microvascular vasomotion and meliorates iECs function due to anti-hyperglycemic and anti-oxidative effects, partly through mechanism involving regulation of eNOS and p-eNOS.

Article	Year
Experimental study on the function of cardiomyocytes. Clinical hemorheology and microcirculation\| PubMed ID: 14724363	2003
The mitogenic and anti-apoptotic activity of tumor conditioned medium on endothelium. Clinical hemorheology and microcirculation\| PubMed ID: 14724364	2003
A proteomic study on cell cycle progression of endothelium exposed to tumor conditioned medium and the possible role of cyclin D1/E. Clinical hemorheology and microcirculation\| PubMed ID: 14724365	2003
Effect and mechanism of melatonin's action on the proliferation of human umbilical vein endothelial cells. Journal of pineal research\| PubMed ID: 17014693	2006
Peroxisome proliferator-activated receptor-gamma ligands 15-deoxy-delta(12,14)-prostaglandin J2 and pioglitazone inhibit hydroxyl peroxide-induced TNF-alpha and lipopolysaccharide-induced CX... Shock (Augusta, Ga.)\| PubMed ID: 19174742	2009
SALL4, a stem cell factor, affects the side population by regulation of the ATP-binding cassette drug transport genes. PloS one\| PubMed ID: 21526180	2011
SALL4 is a new target in endometrial cancer. Oncogene\| PubMed ID: 24336327	2015
Insulin treatment restores islet microvascular vasomotion function in diabetic mice. Journal of diabetes\| PubMed ID: 27976498	2017
BST-2 restricts IAV release and is countered by the viral M2 protein. The Biochemical journal\| PubMed ID: 28087685	2017
Functional status of microvascular vasomotion is impaired in spontaneously hypertensive rat. Scientific reports\| PubMed ID: 29213078	2017

Ailing Li

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