Chishimba Nathan Mowa

Estrogen Deprivation and Replacement Modulate Cerebral Capillary Density with Vascular Expression of Angiogenic Molecules in Middle-Aged Female Rats

The effect of postmenopausal estrogen replacement therapy (ERT) on the risk or severity of cerebrovascular disorders is as yet unclear, and the evidence for flow preservation being a mechanism of estrogen neuroprotection remains elusive. The authors examined whether estrogen-mediated flow-preserving neuroprotective mechanisms, if any, may involve its angiogenic action. This study was conducted using middle-aged (44 weeks) female rats because of the importance of aging in cerebrovascular disease in women. Middle-aged female rats were subjected to sham operation, ovariectomy, or ovariectomy with ERT. The anatomic cerebral capillary morphology showed a significant reduction in the total capillary density in the frontal cortex after ovariectomy. This was associated with marked decreases in protein and gene expression of vascular endothelial growth factor and its angiogenic receptors in cerebral vessels, as demonstrated by immunohistochemistry and in situ hybridization. The expression levels of both estrogen receptor (ER) subtypes, ERα and ERβ, in cerebral vessels were significantly reduced after ovariectomy, but ERβ was more dramatically downregulated as assessed by the ERβ/ERα ratio. These ovariectomy-induced changes were completely prevented by ERT. Vascular endothelial growth factor appears to be a critical regulatory molecule for physiologic cerebral angiogenesis in middle-aged female rats and may play an important role in the flow-preserving neuroprotective action of estrogen through its angiogenic and antiapoptotic properties.

Characterization of regional cerebral blood flow and expression of angiogenic growth factors in the frontal cortex of juvenile male SHRSP and SHR

Attention-deficit/hyperactivity disorder (AD/HD) is a common pediatric behavioral disorder associated with male preponderance and reduction of regional cerebral blood flow (rCBF). However, lack of an appropriate animal model exhibiting appropriate AD/HD symptoms stands in the way of studying mechanism(s) underlying reduced rCBF and male preponderance. Our group has been investigating the suitability of juvenile male stroke-prone spontaneously hypertensive rats (SHRSP), a substrain of the commonly used AD/HD animal model SHR, as a model for AD/HD because, unlike SHR, SHRSP displays cognitive impairment and male preponderance. Our more recent studies revealed alterations in the synthesis of sex steroid hormones and angiogenic factors in the frontal cortex of male SHRSP compared to the genetic control WKY. Based on these observations, the present study utilizes laser-Doppler flowmetry, histochemistry, enzyme immunoassay, immunoblotting, and real-time PCR to characterize and compare the patterns of regional cerebral blood flow and synthesis of angiogenic molecules [basic fibroblast growth factor; nitric oxide synthase isoforms (endothelial, neuronal and inducible); vascular endothelial growth factor (VEGF) and its signaling molecules (VEGF receptors, phosphorylated Akt, endothelial nitric oxide synthase eNOS] between male SHRSP and SHR. Overall, consistent with our previous data showing alteration in VEGF/Akt/NO signaling, there was a marked reduction in the profile of rCBF (35%) and angiogenic factors of SHRSP, compared to age-matched genetic control Wistar-Kyoto rats (WKY) and SHR. We conclude that, unlike SHR, the profiles of rCBF and angiogenic factors in SHRSP are altered in juvenile male. Thus, SHRSP appears to be a more suitable animal model for studying changes in rCBF in AD/HD.

VEGF signaling is disrupted in the hearts of mice lacking estrogen receptor alpha.

Estrogen has widely been credited for cardioprotection in women. However, the exact mechanisms that underlie these beneficial estrogenic effects are not completely understood. Here, we sought to: 1) elucidate estrogens influence on levels of vascular endothelial growth factor (VEGF), a key regulator of cardiovascular processes, and components of its basic signaling machinery (VEGF receptors, Akt, and eNOS) in the heart, and 2) delineate the specific estrogen receptor signaling pathway that mediates its beneficial effects using mice lacking either estrogen receptor alpha or estrogen receptor beta. We analyzed pattern of VEGF signaling and the associated coronary capillary density in the hearts of wild-type (WT), estrogen receptor alpha knockout (ERalpha-KO), and estrogen receptor beta knockout (ERbeta-KO) female mice. Deletion of estrogen receptor alpha causes a marked decrease in coronary capillary density compared to wild-type (WT) mice, while that of estrogen receptor beta had a minimal effect. Consistent with reduced coronary capillary density, cardiac expression levels of VEGF and its signaling molecules (two receptors, phosphorylated Akt, and eNOS) in ERalpha-KO mice were reduced to half of WT, in contrast to ERbeta-KO mice that only showed a slight decrease. Moreover, activity of eNOS was greatly lowered in ERalpha-KO mice. These data suggest that estrogen acts largely via estrogen receptor alpha to regulate VEGF transcription and possibly components of its basic signaling and ultimately, the development of coronary microvasculature in the heart. This molecular and histological data, in part, sheds some insights into potential mechanisms that may likely underlie estrogens cardioprotective effects.

Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

BackgroundVEGF-regulated genes in the cervices of pregnant and non-pregnant rodents (rats and mice) were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein.MethodsTissues were analyzed by genome-wide DNA microarray analysis, Real-time and gel-based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray) and ANOVA (Real Time PCR) followed by Scheffes F-test for multiple comparisons.ResultsOf the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF, i.e., expression of about 2,400 and 1,700 genes were down- and up-regulated, respectively. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular- and non-vascular-related processes were found to be regulated by VEGF in the cervix, including immune response (including inflammatory), cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes, known to or with potential to influence cervical remodeling were altered. For example, real time PCR analysis showed that levels of VCAM-1, a key molecule in leukocyte recruitment, endothelial adhesion, and subsequent trans-endothelial migration, were elevated about 10 folds by VEGF. Further, VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. Of note, we show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM.ConclusionThese data are important in that they shed new insights in VEGFs possible roles and mechanisms in cervical events near-term, including cervical remodeling.

Effects of protease activated receptor (PAR)2 blocking peptide on endothelin-1 levels in kidney tissues in endotoxemic rat mode

AIMS Septic shock, the severe form of sepsis, is associated with development of progressive damage in multiple organs. Kidney can be injured and its functions altered by activation of coagulation, vasoactive-peptide and inflammatory processes in sepsis. Endothelin (ET)-1, a potent vasoconstrictor, is implicated in the pathogenesis of sepsis and its complications. Protease-activated receptors (PARs) are shown to play an important role in the interplay between inflammation and coagulation. We examined the time-dependent alterations of ET-1 and inflammatory cytokine, such as tumor necrosis factor (TNF)-α in kidney tissue in lipopolysaccharide (LPS)-induced septic rat model and the effects of PAR2 blocking peptide on the LPS-induced elevations of renal ET-1 and TNF-α levels. MAIN METHODS Male Wistar rats at 8 weeks of age were administered with either saline solution or LPS at different time points (1, 3, 6 and 10h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide for 3h to assess whether blockade of PAR2 has a regulatory role on the ET-1 level in septic kidney. KEY FINDINGS An increase in ET-1 peptide level was observed in kidney tissue after LPS administration time-dependently. Levels of renal TNF-α peaked (around 12-fold) at 1h of sepsis. Interestingly, PAR2 blocking peptide normalized the LPS-induced elevations of renal ET-1 and TNF-α levels. SIGNIFICANCE The present study reveals a distinct chronological expression of ET-1 and TNF-α in LPS-administered renal tissues and that blockade of PAR2 may play a crucial role in treating renal injury, via normalization of inflammation, coagulation and vaso-active peptide.

Disruption of components of vascular endothelial growth factor angiogenic signalling system in metabolic syndrome. Findings from a study conducted in rural Bangladeshi women.

Metabolic syndrome (MetS) is associated with impaired angiogenesis, a process that is chiefly regulated by vascular endothelial growth factor (VEGF) upon binding to its specific receptors, VEGF-R1 and VEGF-R2. The purpose of the present study was to assess trends or patterns in plasma levels of VEGF and its soluble receptors in subjects with (MetS) or without (non-MetS) MetS; and further examine their association with clinical or metabolic parameters using a subpopulation of South Asian country. A total of 1,802 rural Bangladeshi women aged ≥15 years were studied using a population-based cross-sectional survey. Plasma levels of VEGF were found to be significantly increased (MetS vs. non-MetS: 483.9 vs. 386.9, p<0.001), whereas, the soluble forms of VEGF receptors, sVEGF-R1 and sVEGF-R2, were significantly decreased in subjects with Mets (sVEGF-R1, MetS vs. non-MetS: 512.5 vs. 631.3, p<0.001; sVEGF-R2, MetS vs. non-MetS: 9,302.8 vs. 9,787.4, p=0.004). After adjustment for age and all potential variables, multiple regression analysis revealed that plasma levels of VEGF had significant positive association with blood glucose (p = 0.019) and body mass index (p = 0.007). We also found that mean plasma levels of VEGF increased in direct proportion to levels of MetS components. The present study is the first ever to demonstrate a positive association between trends in levels of plasma VEGF and MetS using a large sample size from South Asia. The association between plasma VEGF and MetS needs further investigations in order to clearly decipher the clinical predictive value and accuracy of plasma VEGF in MetS.

Vascular endothelial growth factor induces growth of uterine cervix and immune cell recruitment in mice

Knowledge of uterine cervical epithelial biology and factors that influence its events may be critical in understanding the process of cervical remodeling (CR). Here, we examine the impact of exogenous vascular endothelial growth factor (VEGF) on uterine cervical epithelial growth in mice (nonpregnant and pregnant) treated with VEGF agents (recombinant and inhibitor) using a variety of morphological and molecular techniques. Exogenous VEGF altered various uterine cervical epithelial cellular events, including marked induction of growth, edema, increase in inter-epithelial paracellular space, and recruitment of immune cells to the outer surface of epithelial cells (cervical lumen). We conclude that VEGF induces multiple alterations in the uterine cervical epithelial tissues that may play a role in local immune surveillance and uterine cervical growth during CR.

Postpartum cervical repair in mice: a morphological characterization and potential role for angiogenic factors

The cervix undergoes marked mechanical trauma during delivery of the baby at birth. As such, a timely and complete tissue repair postpartum is necessary to prevent obstetrical complications, such as cervicitis, ectropion, hemorrhage, repeated miscarriages or abortions and possibly preterm labor and malignancies. However, our knowledge of normal cervical repair is currently incomplete and factors that influence repair are unclear. Here, we characterize the morphological and angiogenic profile of postpartum repair in mice cervix during the first 48 h of postpartum. The key findings presented here are: (1) cervical epithelial folds and size are diminished during the first 48 h of postpartum repair, (2) hypoxic inducible factor 1a, vascular endothelial growth factor (VEGF), and VEGF receptor 1 expression are pronounced early in postpartum cervical repair, and (3) VEGF receptor 2 gene and protein expressions are variable. We conclude that postpartum cervical repair involves gross and microscopic changes and is linked to expression of angiogenic factors. Future studies will assess the suitability of these factors, identified in the present study, as potential markers for determining the phase of postpartum cervical repair in obstetrical complications, such as cervical lacerations.

Landiolol hydrochloride ameliorates acute lung injury in a rat model of early sepsis through the suppression of elevated levels of pulmonary endothelin-1

Among the dysfunctions and pathologies associated with sepsis, the underlying molecular mechanisms of sepsis-induced acute lung injury (ALI) are poorly understood. Endothelin (ET)-1, a potent vasoconstrictor and pro-inflammatory peptide, is known to be involved in the pathogenesis of ALI in a rat model of sepsis. Here, we investigated whether landiolol hydrochloride, an ultra-short-acting β-blocker, plays a crucial role in ameliorating and attenuating LPS-induced ALI through modulation of the ET-1 system. Male Wistar rats at 8weeks of age were administered with either saline or lipopolysaccharide (LPS) for three hours (3h) and some of the LPS-administered rats were continuously treated with landiolol for 3h. ALI was induced by LPS, including levels of both circulatory and pulmonary TNF-α and IL-6 but [PaO2] was significantly decreased. LPS also induced a significant increase in levels of pulmonary ET-1 and ET-A receptor, but levels of ET-B receptor, which has vasodilating effects, were remarkably diminished. Further, LPS administration upregulated the pulmonary expression of HIF-1α. Finally, the treatment of LPS-administered rats with landiolol for 3h ameliorated and prevented ALI, normalized the altered levels of pulmonary ET-1 and ET-A receptors. Landiolol also induced significant down-regulation of ET-B receptor in lung tissues in the early hours (phase) of sepsis. However, Landiolol treatment had no effect on the up-regulated inflammatory mediators (TNF-α, IL-6) in both plasma and lung tissues during sepsis, and expression of pulmonary HIF-1α also remained unchanged after landiolol treatment. Collectively, these data led us to conclude that landiolol may ameliorate sepsis-induced ALI via the pulmonary ET system.

Dual blockade of endothelin action exacerbates up-regulated VEGF angiogenic signaling in the heart of lipopolysaccharide-induced endotoxemic rat model

AIMS Sepsis is a cluster of heterogeneous syndromes associated with progressive endotoxemic developments, ultimately leading to damage of multiple organs, including the heart. However, the pathogenesis of sepsis-induced myocardial dysfunction is still not fully understood. The present study is the first to examine alterations in expression of key angiogenic signaling system mediated by vascular endothelial growth factor (VEGF) in septic heart and the effects of endothelin dual blocker (ETDB) on it. MAIN METHODS Normal Wistar rats were either administered with: a) vehicle only (control group), b) lipopolysaccharide only (LPS: 15 mg/kg) and then sacrificed at different time points (1 h, 3 h, 6 h and 10 h), and c) the last group was co-administered with LPS and ETDB (SB-209670, 1 mg/kg body weight) for 6 h and then sacrificed. KEY FINDINGS Administration of LPS resulted in increases in levels of: a) serum tumor necrosis factor (TNF)-α, b) serum VEGF and c) serum endothelin (ET)-1 levels accompanied by up-regulation of cardiac VEGF and its downstream angiogenic signaling molecules. While cardiac TNF-α level was unchanged among experimental groups, cardiac ET-1 level was significantly higher in LPS-administered group. SIGNIFICANCE We conclude that elevation in VEGF angiogenic signaling may be triggered by diminished oxygenation in the myocardium following LPS administration as a consequence of sepsis-induced microvascular dysfunction. Because of this cardiac dysfunction, oxygen supply may be inadequate at microregional level to support the normal heart metabolism and function. ETDB at 6 h further increased the elevated levels of VEGF angiogenic signaling in endotoxemic heart.