Wenhong Luo

Urine formaldehyde level is inversely correlated to mini mental state examination scores in senile dementia

It is widely known that exogenous formaldehyde exposure induces human cognitive impairment and animal memory loss; and recent studies show that formaldehyde at pathological levels induces Aβ deposition and misfolding of tau protein to form globular amyloid-like aggregates. Endogenous formaldehyde may be a marker for progressive senile dementia. The aim of this study was to investigate the correlation of endogenous formaldehyde in urine of senile dementia and mini mental state examination (MMSE) scores. Formaldehyde level was analyzed by high-performance liquid chromatography (with fluorescence detection) in human urine from dementia patients (n=141), patients with hypertension (n=33) or diabetes (n=16) and healthy individuals (n=38), autopsy hippocampus samples from Alzheimers disease (AD) patients and brains of three types of AD animal model: namely, senescence accelerated mice (SAMP8), APP-transgenic mice and APP/PS1-transgenic mice. In a double-blind study, there was marked elevation of urine formaldehyde levels in patients (n=91) with dementia, and a slight increase in patients (n=50) with mild cognitive impairment. Urine formaldehyde level was inversely correlated with mini mental state examination scores (Rs=-0.441, p<0.0001). Furthermore, formaldehyde levels were significantly increased in the autopsy hippocampus from Alzheimers patients (n=4). In SAMP8 brains the formaldehyde level was significantly increased, suggesting that the endogenous formaldehyde is related to aging in mice. The brain formaldehyde level in APP/PS1-transgenic (n=8) mice at age of 3 months and APP-transgenic (n=8) mice at age of 6 months was increased (0.56 ± 0.02 mM), respectively, as compared with their respective age-matched controls, when these two types of AD-like animals, respectively, started to form Aβ deposits and memory loss obviously. According to the level of formaldehyde in the brain of the transgenic mice, we treated normal mice with formaldehyde (0.5mM, intraperitoneal administration) and observed the memory loss of the animal in Morris water maze trial. Cognitive impairments for the senile dementia are probably related to endogenous formaldehyde levels; and the mini mental state examination scores referred to the evaluation of urine formaldehyde level in dementia patients may be used as a non-invasive method for the investigation and diagnosis of senile dementia.

Tumor Tissue-Derived Formaldehyde and Acidic Microenvironment Synergistically Induce Bone Cancer Pain

Background There is current interest in understanding the molecular mechanisms of tumor-induced bone pain. Accumulated evidence shows that endogenous formaldehyde concentrations are elevated in the blood or urine of patients with breast, prostate or bladder cancer. These cancers are frequently associated with cancer pain especially after bone metastasis. It is well known that transient receptor potential vanilloid receptor 1 (TRPV1) participates in cancer pain. The present study aims to demonstrate that the tumor tissue-derived endogenous formaldehyde induces bone cancer pain via TRPV1 activation under tumor acidic environment. Methodology/Principal Findings Endogenous formaldehyde concentration increased significantly in the cultured breast cancer cell lines in vitro, in the bone marrow of breast MRMT-1 bone cancer pain model in rats and in tissues from breast cancer and lung cancer patients in vivo. Low concentrations (1∼5 mM) of formaldehyde induced pain responses in rat via TRPV1 and this pain response could be significantly enhanced by pH 6.0 (mimicking the acidic tumor microenvironment). Formaldehyde at low concentrations (1 mM to 100 mM) induced a concentration-dependent increase of [Ca2+]i in the freshly isolated rat dorsal root ganglion neurons and TRPV1-transfected CHO cells. Furthermore, electrophysiological experiments showed that low concentration formaldehyde-elicited TRPV1 currents could be significantly potentiated by low pH (6.0). TRPV1 antagonists and formaldehyde scavengers attenuated bone cancer pain responses. Conclusions/Significance Our data suggest that cancer tissues directly secrete endogenous formaldehyde, and this formaldehyde at low concentration induces metastatic bone cancer pain through TRPV1 activation especially under tumor acidic environment.

Aging-associated excess formaldehyde leads to spatial memory deficits

Recent studies show that formaldehyde participates in DNA demethylation/methylation cycle. Emerging evidence identifies that neuronal activity induces global DNA demethylation and re-methylation; and DNA methylation is a critical step for memory formation. These data suggest that endogenous formaldehyde may intrinsically link learning-responsive DNA methylation status and memory formation. Here, we report that during spatial memory formation process, spatial training induces an initial global DNA demethylation and subsequent re-methylation associated with hippocampal formaldehyde elevation then decline to baseline level in Sprague Dawley rats. Scavenging this elevated formaldehyde by formaldehyde-degrading enzyme (FDH), or enhancing DNA demethylation by a DNA demethylating agent, both led to spatial memory deficits by blocking DNA re-methylation in rats. Furthermore, we found that the normal adult rats intrahippocampally injected with excess formaldehyde can imitate the aged-related spatial memory deficits and global DNA methylation decline. These findings indicate that aging-associated excess formaldheyde contributes to cognitive decline during aging.

Involvement of cyclo‐oxygenase‐1‐mediated prostacyclin synthesis in the vasoconstrictor activity evoked by ACh in mouse arteries

This study was to determine whether the endothelium of mouse major arteries produces prostacyclin (PGI2) and, if so, to determine how PGI2 affects vasomotor reactivity and whether cyclo‐oxygenase‐1 (COX‐1) contributes to PGI2 synthesis. Abdominal aortas, carotid and femoral arteries were isolated from wild‐type mice and/or those with COX‐1 or ‐2 deficiency (COX‐1−/−; COX‐2−/−) for biochemical and/or functional analyses. The PGI2 metabolite 6‐keto‐PGF1α was analysed with high‐performance liquid chromatography–mass spectroscopy, while vasoreactivity was determined with isometric force measurement. Results showed that in the abdominal aorta, ACh evoked endothelium‐dependent production of 6‐keto‐PGF1α, which was abolished by COX‐1−/−, but not by COX‐2−/−. Interestingly, COX‐1−/− enhanced the dilatation in response to ACh, while PGI2, which evoked relaxation of the mesenteric artery, caused contraction that was abolished by antagonizing thromboxane prostanoid (TP) receptors in the abdominal aorta. However, the TP receptor agonist U46619 evoked similar contractions in the abdominal aorta and mesenteric artery. Also, antagonizing TP receptors enhanced the relaxation in response to PGI2 in mesenteric arteries. Real‐time PCR showed that the PGI2 (IP) receptor mRNA level was lower in the abdominal aorta than in mesenteric arteries. In addition, COX‐1−/− not only abolished the contraction in response to ACh following NO inhibition in abdominal aorta, but also those in the carotid and femoral arteries. These results demonstrate an explicit role for endothelial COX‐1 in PGI2 synthesis and suggest that in given mouse arteries, PGI2 mediates not dilatation but rather vasoconstrictor activity, possibly due to a low expression or functional presence of IP receptors, which enables PGI2 to act mainly on TP receptors.

Age-related formaldehyde interferes with DNA methyltransferase function, causing memory loss in Alzheimer's disease

Hippocampus-related topographic amnesia is the most common symptom of memory disorders in Alzheimers disease (AD) patients. Recent studies have revealed that experience-mediated DNA methylation, which is regulated by enzymes with DNA methyltransferase (DNMT) activity, is required for the formation of recent memory as well as the maintenance of remote memory. Notably, overexpression of DNMT3a in the hippocampus can reverse spatial memory deficits in aged mice. However, a decline in global DNA methylation was found in the autopsied hippocampi of patients with AD. Exactly, what endogenous factors that affect DNA methylation still remain to be elucidated. Here, we report a marked increase in endogenous formaldehyde levels is associated with a decline in global DNA methylation in the autopsied hippocampus from AD patients. In vitro and in vivo results show that formaldehyde in excess of normal physiological levels reduced global DNA methylation by interfering DNMTs. Interestingly, intrahippocampal injection of excess formaldehyde before spatial learning in healthy adult rats can mimic the learning difficulty of early stage of AD. Moreover, injection of excess formaldehyde after spatial learning can mimic the loss of remote spatial memory observed in late stage of AD. These findings suggest that aging-associated formaldehyde contributes to topographic amnesia in AD patients.

Lysine-Specific Demethylase 1 in Breast Cancer Cells Contributes to the Production of Endogenous Formaldehyde in the Metastatic Bone Cancer Pain Model of Rats

Background Bone cancer pain seriously affects the quality of life of cancer patients. Our previous study found that endogenous formaldehyde was produced by cancer cells metastasized into bone marrows and played an important role in bone cancer pain. However, the mechanism of production of this endogenous formaldehyde by metastatic cancer cells was unknown in bone cancer pain rats. Lysine-specific demethylase 1 (LSD1) is one of the major enzymes catalyzing the production of formaldehyde. The expression of LSD1 and the concentration of formaldehyde were up-regulated in many high-risk tumors. Objective This study aimed to investigate whether LSD1 in metastasized MRMT-1 breast cancer cells in bone marrows participated in the production of endogenous formaldehyde in bone cancer pain rats. Methodology/Principal Findings Concentration of the endogenous formaldehyde was measured by high performance liquid chromatography (HPLC). Endogenous formaldehyde dramatically increased in cultured MRMT-1 breast cancer cells in vitro, in bone marrows and sera of bone cancer pain rats, in tumor tissues and sera of MRMT-1 subcutaneous vaccination model rats in vivo. Formaldehyde at a concentration as low as the above measured (3 mM) induced pain behaviors in normal rats. The expression of LSD1 which mainly located in nuclei of cancer cells significantly increased in bone marrows of bone cancer pain rats from 14 d to 21 d after inoculation. Furthermore, inhibition of LSD1 decreased the production of formaldehyde in MRMT-1 cells in vitro. Intraperitoneal injection of LSD1 inhibitor pargyline from 3 d to 14 d after inoculation of MRMT-1 cancer cells reduced bone cancer pain behaviors. Conclusion Our data in the present study, combing our previous report, suggested that in the endogenous formaldehyde-induced pain in bone cancer pain rats, LSD1 in metastasized cancer cells contributed to the production of the endogenous formaldehyde.

Concomitant activation of functionally opposing prostacyclin and thromboxane prostanoid receptors by cyclo‐oxygenase‐1‐mediated prostacyclin synthesis in mouse arteries

This study aimed to determine whether cyclo‐oxygenase‐1 (COX‐1) mediates dilatation of mouse arteries via synthesis of prostacyclin (PGI2) and, if so, how PGI2 (IP) receptors contribute and whether thromboxane prostanoid (TP) receptors are implicated in the process. Mesenteric arteries were isolated from wild‐type mice or mice with COX‐1 deficiency (COX‐1−/−). The vasomotor reaction to the COX substrate arachidonic acid (AA) was determined with isometric force measurement, while the in vitro production or the plasma level of the PGI2 metabolite 6‐keto‐PGF1α was analysed with high‐performance liquid chromatography–mass spectroscopy or enzyme immunoassay, respectively. Results showed that AA, which evoked endothelium‐dependent 6‐keto‐PGF1α production, elicited relaxation that was inhibited or enhanced by antagonizing IP or TP receptors, respectively. Also, IP receptor blockade resulted in contraction in response to AA (following NO synthase inhibition), which was prevented by a concomitant TP receptor antagonism. Meanwhile, COX‐1−/− or COX‐1 inhibition abolished the in vitro 6‐keto‐PGF1α production and reduced the relaxation or contraction observed with AA. Real‐time PCR showed that whereas TP receptor mRNAs were detected at similar levels, IP receptor mRNAs were present at higher levels in the branches than in the main stem of the mesenteric artery. In addition, antagonizing the IP receptors enhanced the contraction evoked by PGI2 in the carotid artery. Also, we noted that COX‐1−/− mice had a reduced basal plasma 6‐keto‐PGF1α level. These results demonstrate an explicit vasodilator role for COX‐1‐mediated endothelial PGI2 synthesis and suggest that the functionally opposing IP and TP receptors concomitantly mediate the vasomotor reaction to PGI2, with the dilator activity of IP receptors being compromised by the vasoconstrictor effect of TP receptors and vice versa.

Cyclo‐oxygenase‐1 or ‐2‐mediated metabolism of arachidonic acid in endothelium‐dependent contraction of mouse arteries

•  What is the central question of this study? To determine the specific cyclo‐oxygenase (COX) isoform(s) involved in endothelium‐dependent contraction and whether prostaglandin I2, a mediator of endothelium‐derived vasoconstrictor activity, can be generated in medial smooth muscle from prostaglandin H2 that might diffuse from the endothelium. •  What is the main finding and its importance? Our results demonstrate a predominant role for COX‐1 in arachidonic acid metabolism and suggest that in the given mouse arteries, metabolites from either COX isoform cause contraction. Moreover, our results imply that some of the prostaglandin I2 involved in the vasoconstrictor activity of endothelial COX‐mediated metabolism could possibly be generated from prostaglandin H2 in the medial smooth muscle. These findings add to our current understanding of mechanisms for endothelium‐dependent contraction.

The endothelial cyclooxygenase pathway: Insights from mouse arteries

To date, cyclooxygenase-2 (COX-2) is commonly believed to be the major mediator of endothelial prostacyclin (prostaglandin I2; PGI2) synthesis that balances the effect of thromboxane (Tx) A2 synthesis mediated by the other COX isoform, COX-1 in platelets. Accordingly, selective inhibition of COX-2 is considered to cause vasoconstriction, platelet aggregation, and hence increase the incidence of cardiovascular events. This idea has been claimed to be substantiated by experiments on mouse models, some of which are deficient in one of the two COX isoforms. However, results from our studies and those of others using similar mouse models suggest that COX-1 is the major functional isoform in vascular endothelium. Also, although PGI2 is recognized as a potent vasodilator, in some arteries endothelial COX activation causes vasoconstrictor response. This has again been recognized by studies, especially those performed on mouse arteries, to result largely from endothelial PGI2 synthesis. Therefore, evidence that supports a role for COX-1 as the major mediator of PGI2 synthesis in mouse vascular endothelium, reasons for the inconsistency, and results that elucidate underlying mechanisms for divergent vasomotor reactions to endothelial COX activation will be discussed in this review. In addition, we address the possible pathological implications and limitations of findings obtained from studies performed on mouse arteries.

Role of cyclooxygenase-1 and -2 in endothelium-dependent contraction of atherosclerotic mouse abdominal aortas.

The objective of this study was to determine the role of cyclooxygenase (COX)‐1 or ‐2 in endothelium‐dependent contraction under atherosclerotic conditions. Atherosclerosis was induced in apoE knockout (apoE−/−) mice and those with COX‐1−/− (apoE−/−‐COX‐1−/−) by feeding with high fat and cholesterol food. Aortas (abdominal or the whole section) were isolated for functional and/or biochemical analyses. As in non‐atherosclerotic conditions, the muscarinic receptor agonist acetylcholine (ACh) evoked an endothelium‐dependent, COX‐mediated contraction following NO synthase (NOS) inhibition in abdominal aortic rings from atherosclerotic apoE−/− mice. Interestingly, COX‐1 inhibition not only abolished such a contraction in rings showing normal appearance, but also diminished that in rings with plaques. Accordingly, only a minor contraction (<30% that of apoE−/− counterparts) was evoked by ACh (following NOS inhibition) in abdominal aortic rings of atherosclerotic apoE−/−‐COX‐1−/− mice with plaques, and none was evoked in those showing normal appearance. Also, the contraction evoked by ACh in apoE−/−‐COX‐1−/− abdominal aortic rings with plaques was abolished by non‐selective COX inhibition, thromboxane‐prostanoid (TP) receptor antagonism, or endothelial denudation. Moreover, it was noted that ACh evoked a predominant production of the prostacyclin (PGI2, which mediates abdominal aortic contraction via TP receptors in mice) metabolite 6‐keto‐PGF1α, which was again sensitive to COX‐1 inhibition or COX‐1−/−. Therefore, in atherosclerotic mouse abdominal aortas, COX‐1 can still be the major isoform mediating endothelium‐dependent contraction, which probably results largely from PGI2 synthesis as in non‐atherosclerotic conditions. In contrast, COX‐2 may have only a minor role in such response limited to areas of plaques under the same pathological condition.