Darrell Sawmiller

Vasoactive intestinal peptide: cardiovascular effects

Vasoactive intestinal peptide (VIP) is present in the peripheral and the central nervous systems where it functions as a nonadrenergic, noncholinergic neurotransmitter or neuromodulator. Significant concentrations of VIP are present in the gastrointestinal tract, heart, lungs, thyroid, kidney, urinary bladder, genital organs and the brain. On a molar basis, VIP is 50-100 times more potent than acetylcholine as a vasodilator. VIP release in the body is stimulated by high frequency (10-20 Hz) nerve stimulation and by cholinergic agonists, serotonin, dopaminergic agonists, prostaglandins (PGE, PGD), and nerve growth factor. The VIP peptide combines with its receptor and dose-dependently activates adenylyl cyclase. The vasodilatory effect of VIP in different vascular tissues or species also may be due to increases in nitric oxide, cyclic GMP, and other signaling agents. In the heart, VIP immunoreactive nerve fibers are present not only in the epicardial coronary arteries and veins, but also the sinoatrial node, atrium, interatrial septum, atrioventricular node, intracardiac ganglia, and ventricles (right ventricle >> left ventricle). In the coronary arterial walls, VIP may contribute to the regulation of normal coronary vasomotor tone. In research animals and in humans, VIP, administered into the coronary artery or intravenously, increases the epicardial coronary artery cross-sectional area, decreases coronary vascular resistance, and significantly increases coronary artery blood flow. High frequency parasympathetic (vagal) nerve stimulation also releases endogenous VIP in the coronary vessels and heart and significantly increases coronary artery blood flow. In addition, the release of VIP in the heart is increased during coronary artery occlusion and during reperfusion where VIP may promote local blood flow and may have a free-radical scavenging effect. VIP also has a primary positive inotropic effect on cardiac muscle that is enhanced by its ability to facilitate ventricular-vascular coupling by reducing mean arterial pressure by 10-15%. In concentrations of 10(-8)-10(-5) mol, VIP augments developed isometric force and increases atrial and ventricular contractility. The presence of VIP-immunoreactive nerve fibers in and around the sinus and the atrioventricular nodes of mammals strongly suggests that this peptide can affect the heart rate. In this regard, endogenously released or exogenous VIP can significantly increase the heart rate and has a more potent effect on heart rate than does norepinephrine. The presence and significant cardiovascular effects of VIP in the heart suggests that this peptide is important in the regulation of coronary blood flow, cardiac contraction, and heart rate. Current investigations are defining the physiological role of VIP in the regulation of cardiovascular function.

Luteolin Reduces Alzheimer’s Disease Pathologies Induced by Traumatic Brain Injury

Traumatic brain injury (TBI) occurs in response to an acute insult to the head and is recognized as a major risk factor for Alzheimer’s disease (AD). Indeed, recent studies have suggested a pathological overlap between TBI and AD, with both conditions exhibiting amyloid-beta (Aβ) deposits, tauopathy, and neuroinflammation. Additional studies involving animal models of AD indicate that some AD-related genotypic determinants may be critical factors enhancing temporal and phenotypic symptoms of TBI. Thus in the present study, we examined sub-acute effects of moderate TBI delivered by a gas-driven shock tube device in Aβ depositing Tg2576 mice. Three days later, significant increases in b-amyloid deposition, glycogen synthase-3 (GSK-3) activation, phospho-tau, and pro-inflammatory cytokines were observed. Importantly, peripheral treatment with the naturally occurring flavonoid, luteolin, significantly abolished these accelerated pathologies. This study lays the groundwork for a safe and natural compound that could prevent or treat TBI with minimal or no deleterious side effects in combat personnel and others at risk or who have experienced TBI.

Baicalein reduces β‐amyloid and promotes nonamyloidogenic amyloid precursor protein processing in an Alzheimer's disease transgenic mouse model

Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ‐aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABAA modulation for Alzheimers disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild‐type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of β‐amyloid (Aβ) by increasing APP α‐processing. These effects were blocked by the GABAA antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α‐secretase processing, reduced Aβ production, and reduced AD‐like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aβ production and improving cognitive performance, by activating GABAA receptors.

Soluble amyloid precursor protein alpha inhibits tau phosphorylation through modulation of GSK3β signaling pathway.

We recently found that sAPPα decreases amyloid‐beta generation by directly associating with β‐site amyloid precursor protein (APP)‐converting enzyme 1 (BACE1), thereby modulating APP processing. Because inhibition of BACE1 decreases glycogen synthase kinase 3 beta (GSK3β)‐mediated Alzheimers disease (AD)‐like tau phosphorylation in AD patient‐derived neurons, we determined whether sAPPα also reduces GSK3β‐mediated tau phosphorylation. We initially found increased levels of inhibitory phosphorylation of GSK3β (Ser9) in primary neurons from sAPPα over‐expressing mice. Further, recombinant human sAPPα evoked the same phenomenon in SH‐SY5Y cells. Further, in SH‐SY5Y cells over‐expressing BACE1, and HeLa cells over‐expressing human tau, sAPPα reduced GSK3β activity and tau phosphorylation. Importantly, the reductions in GSK3β activity and tau phosphorylation elicited by sAPPα were prevented by BACE1 but not γ‐secretase inhibition. In accord, AD mice over‐expressing human sAPPα had less GSK3β activity and tau phosphorylation compared with controls. These results implicate a direct relationship between APP β‐processing and GSK3β‐mediated tau phosphorylation and further define the central role of sAPPα in APP autoregulation and AD pathogenesis.

Human umbilical cord blood mononuclear cells activate the survival protein Akt in cardiac myocytes and endothelial cells that limits apoptosis and necrosis during hypoxia

We have previously reported that human umbilical cord blood mononuclear cells (HUCBC), which contain hematopoietic, mesenchymal, and endothelial stem cells, can significantly reduce acute myocardial infarction size. To determine the mechanism whereby HUCBC increase myocyte and vascular endothelial cell survival, we treated cardiac myocytes and coronary artery endothelial cells in separate experiments with HUCBC plus culture media or culture media alone and subjected the cells to 24 h of hypoxia or normoxia. We then determined in myocytes and endothelial cells activation of the cell survival protein Akt by Western blots. We also determined in these cells apoptosis by annexin V staining and necrosis by propidium iodide staining. Thereafter, we inhibited with API, a specific and sensitive Akt inhibitor, Akt activation in myocytes and endothelial cells cultured with HUCBC during hypoxia and determined cell apoptosis and necrosis. In cells cultured without HUCBC, hypoxia only slightly activated Akt. Moreover, hypoxia increased myocyte apoptosis by ≥ 226% and necrosis by 58% in comparison with myocytes in normoxia. Hypoxic treatment of endothelial cells without HUCBC increased apoptosis by 94% and necrosis by 59%. In contrast, hypoxia did not significantly affect HUCBC. Moreover, in myocyte + HUCBC cultures in hypoxia, HUCBC induced a ≥ 135% increase in myocyte phospho-Akt. Akt activation decreased myocyte apoptosis by 76% and necrosis by 35%. In endothelial cells, HUCBC increased phospho-Akt by 116%. HUCBC also decreased endothelial cell apoptosis by 58% and necrosis by 42%. Inhibition of Akt with API in myocytes and endothelial cells cultured with HUCBC during hypoxia nearly totally prevented the HUCBC-induced decrease in apoptosis and necrosis. We conclude that HUCBC can significantly decrease hypoxia-induced myocyte and endothelial cell apoptosis and necrosis by activating Akt in these cells and in this manner HUCBC can limit myocardial ischemia and injury.

Restoring Soluble Amyloid Precursor Protein α Functions as a Potential Treatment for Alzheimer's Disease

Soluble amyloid precursor protein α (sAPPα), a secreted proteolytic fragment of nonamyloidogenic amyloid precursor protein (APP) processing, is known for numerous neuroprotective functions. These functions include but are not limited to proliferation, neuroprotection, synaptic plasticity, memory formation, neurogenesis, and neuritogenesis in cell culture and animal models. In addition, sAPPα influences amyloid‐β (Aβ) production by direct modulation of APP β‐secretase proteolysis as well as Aβ‐related or unrelated tau pathology, hallmark pathologies of Alzheimers disease (AD). Thus, the restoration of sAPPα levels and functions in the brain by increasing nonamyloidogenic APP processing and/or manipulation of its signaling could reduce AD pathology and cognitive impairment. It is likely that identification and characterization of sAPPα receptors in the brain, downstream effectors, and signaling pathways will pave the way for an attractive therapeutic target for AD prevention or intervention.

Octyl Gallate Markedly Promotes Anti-Amyloidogenic Processing of APP through Estrogen Receptor-Mediated ADAM10 Activation

Our previous studies showed that the green tea-derived polyphenolic compound (−)-epigallocatechin-3 gallate (EGCG) reduces amyloid-β (Aβ) production in both neuronal and mouse Alzheimer’s disease (AD) models in concert with activation of estrogen receptor-α/phosphatidylinositide 3-kinase/protein kinase B (ERα/PI3K/Akt) signaling and anti-amyloidogenic amyloid precursor protein (APP) α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10) processing. Since the gallate moiety in EGCG may correspond to the 7α position of estrogen, thereby facilitating ER binding, we extensively screened the effect of other gallate containing phenolic compounds on APP anti-amyloidogenic processing. Octyl gallate (OG; 10 µM), drastically decreased Aβ generation, in concert with increased APP α-proteolysis, in murine neuron-like cells transfected with human wild-type APP or “Swedish” mutant APP. OG markedly increased production of the neuroprotective amino-terminal APP cleavage product, soluble APP-α (sAPPα). In accord with our previous study, these cleavage events were associated with increased ADAM10 maturation and reduced by blockade of ERα/PI3k/Akt signaling. To validate these findings in vivo, we treated Aβ-overproducing Tg2576 mice with OG daily for one week by intracerebroventricular injection and found decreased Aβ levels associated with increased sAPPα. These data indicate that OG increases anti-amyloidogenic APP α-secretase processing by activation of ERα/PI3k/Akt signaling and ADAM10, suggesting that this compound may be an effective treatment for AD.

Human umbilical cord blood mononuclear cells decrease fibrosis and increase cardiac function in cardiomyopathy

AIMS We investigated whether human umbilical cord blood mononuclear cells (HUCBC) can limit progressive cardiomyopathy in TO2 hamsters. MATERIALS & METHODS A total of 22 TO2 1-month-old hamsters were treated with intramyocardial HUCBC, 4 x 10(6) in Isolyte, and 23 TO2 1-month-old hamsters were treated with intramyocardial Isolyte. A total of 16 1-month-old F1B hamsters served as controls and received intramyocardial Isolyte. Echocardiograms were performed on all hamsters prior to and monthly after treatment for 6 months. Heart tissues were then stained with hematoxylin and eosin, Massons Trichrome and human leukocyte antibody. RESULTS In F1B hamsters, left ventricular fractional shortening (FS) and ejection fractions (EF) did not significantly decrease over 6 months. By contrast, in Isolyte-treated TO2 hamsters, FS decreased from 56.2 +/- 1.0% to 19.7 +/- 3.2% and EF decreased from 89.5 +/- 1.4% to 44.9 +/- 5.9% at 6 months (both p < 0.0001). The FS and EF in HUCBC-treated TO2 hamsters also progressively decreased over 6 months but the changes were more gradual, especially during the first month after HUCBC treatment when FS was 52.0 +/- 1.5% and EF was 89.5 +/- 1.4%, which was not significantly different from the FS and EF in the F1B hamsters. Moreover, in the HUCBC-treated hamsters, the FS and EF were 20-30% greater than FS and EF in Isolyte TO2 hamsters at 3 and 5 months (p < 0.01). In Isolyte-treated TO2 hamsters at 6-7 months, fibrosis involved 30.0 +/- 5.0% of left ventricle and 35.0 +/- 5.0% of septum. By contrast, in HUCBC-treated hamsters, fibrosis involved only 6.5 +/- 2.3% of the left ventricle and 6.3 +/- 1.8% of septum (p < 0.05). The average number of blood vessels per myocardial microscopic field in HUCBC-treated hearts was 53.5 +/- 0.8 versus 46.2 +/- 3.0 in Isolyte-treated TO2 hearts (p < 0.05). CONCLUSION HUCBC, when given as a single intramyocardial injection, can limit fibrosis and increase heart function over the short term in TO2 hamsters with cardiomyopathy.

Coronary vascular effects of vasoactive intestinal peptide in the isolated perfused rat heart

The potency and mechanism of action of vasoactive intestinal peptide (VIP) for producing coronary vasodilation was investigated in the isolated perfused heart of the rat. VIP reduced coronary vascular resistance in a dose-dependent manner, starting at 1 x 10(-10) M, and maximally reduced coronary vascular resistance by 49% at 1 x 10(-8) M. The potency of VIP for reducing coronary vascular resistance (EC50=3.02 x 10(-10) M) was considerably greater than that of adenosine (EC50=6.17 x 10(-8) M) and sodium nitroprusside (EC50=2.45 x 10(-6) M). The vasodilatory action of VIP was more easily observed after increasing vascular tone by perfusion of the hearts with a modified physiological solution containing reduced concentrations of potassium (3.2 mM) and calcium (1.2 mM). Under these conditions, VIP maximally reduced coronary resistance by 54% at 7 x 10(-9) M. The potency of VIP for reducing coronary resistance in these hearts, however, decreased 16-fold (EC50=4.90 x 10(-9) M) while that of SNP remained unaltered (EC50=3.39 x 10(-6) M), compared with hearts perfused with higher levels of potassium (5.9 mM) and calcium (2.5 mM). The vasodilatory effect of VIP occurred without a significant change in heart rate, myocardial contractility or oxygen consumption. In additional studies, the dose-dependent effect of VIP on cyclic nucleotide release from the heart was determined by infusing VIP into the coronary circulation in a cumulative fashion to produce final concentrations between 1 x 10(-11) and 1 x 10(-9) M. VIP increased cyclic AMP at 1 x 10(-9) M but did not increase cyclic GMP. Studies using RT-PCR and immunohistochemistry clearly demonstrated the presence of two VIP receptor subtypes, VPAC1 and VPAC2, in the arteries and arterioles of the heart. In conclusion, VIP is a potent vasodilator in the coronary circulation of the rat and the role of VIP in the control of coronary vascular resistance depends on the circulating levels of potassium and calcium. This vasodilatory effect involves binding to specific coronary cell surface receptors, VPAC1 and/or VPAC2, and is dependent on cyclic AMP only during maximal vasodilation.

Human umbilical cord blood-derived monocytes improve cognitive deficits and reduce amyloid-β pathology in PSAPP mice.

Alzheimers disease (AD) is the fourth major cause of mortality in the elderly in the US and the leading cause of dementia worldwide. While pharmacological targets have been discovered, there are no true disease-modifying therapies. We have recently discovered that multiple low-dose infusions of human umbilical cord blood cells (HUCBCs) ameliorate cognitive impairments and reduce Aβ-associated neuropathology in PSAPP transgenic mice. However, the mechanism for these effects of HUCBCs remains unclear. In the present study, we examined whether monocytes, as important components of HUCBCs, would have beneficial outcomes on the reduction of AD-like pathology and associated cognitive impairments in PSAPP transgenic AD model mice. PSAPP mice and their wild-type littermates were treated monthly with an infusion of peripheral human umbilical cord blood cell (HUCBC)-derived monocytes over a period of 2 and 4 months, followed by behavioral evaluations, biochemical, and histological analyses. The principal findings of the present study confirmed that monocytes derived from HUCBCs (CB-M) play a central role in HUCBC-mediated cognition-enhancing and Aβ pathology-ameliorating activities. Most importantly, we found that compared with CB-M, aged monocytes showed an ineffective phagocytosis of Aβ, while exogenous soluble amyloid precursor protein α (sAPPα) could reverse this deficiency. Pretreating monocytes with sAPPα upregulates Aβ internalization. Our further studies suggested that sAPPα could form a heterodimer with Aβs, with the APP672-688 (Aβ1-16) region being responsible for this effect. This in turn promoted binding of these heterodimers to monocyte scavenger receptors and thus promoted enhanced Aβ clearance. In summary, our findings suggest an interesting hypothesis that peripheral monocytes contribute to Aβ clearance through heterodimerization of sAPPα with Aβ. Further, declined or impaired sAPPaα production, or reduced heterodimerization with Aβ, would cause a deficiency in Aβ clearance and thus accelerate the pathogenesis of AD.