Angiogenic Growth Factor Flood

A previous series, about p53 culminated with "Blastocyst Development - A Perfected Cancer Model" that focused on the parallels in angiogenesis, triggered by blastocyst implantation and progression of tumors beyond ~1mm. Now, a recent study has found that conventional Natural Killer cells (cNK) control vascular remodeling in the uterus during pregnancy by acidifying the extracellular matrix (ECM) with a2V-ATPase that activates MMP-9 that degrades the ECM. Ablation of a2V-ATPase decreases Bax and p53 expression in testis and leads to implantation failure in the female mouse. The degrading ECM releases bound pro-angiogenic growth factors that contribute to Uterine artery (UtA) remodeling characterized by the loss of vascular smooth muscle cells (VSMCs) and dilation of the vessels. Without cNK, the UtA never lose VSMCs and UtA resistance remains high often leading to implantation failure.

Its logical that a timely flood of angiogenic growth factors, previously stored in the ECM would provide instant availability, but whether this explains the maternal-embryonic immune paradox remains to be determined? In the immune paradox maternal NK cells invade and maternal blood vessels are remodeled just before the arrival of trophoblasts, the external cells of the blastocyst, that carry male antigens during formation of the fetal placenta. A sudden flood of angiogenic factors preceding invading trophoblasts could provide the perfect environment required for maternal arterial/vascular remodeling.

Lymphocytes in the uterine lining (decidua) are dominated by a unique decidual natural killer (dNK) cell population. The dNK cell surface phenotype CD56bright CD16− CD3− and macrophages CD14+ CD206+(dMac) support a model whereby dNK cells, capable of killing extra-villous cytotrophoblasts (CTB), are prevented from doing so by neighboring macrophages thus protecting the fetal cells from NK cell attack. Existing research has centered on the function of the abundant and diverse sets of dNK, but now that cNK cells have been identified to play a more significant role, our understanding of the remodeling are likely to change.

In CTB exogenous p53 is able to down-regulate MMP-9 promoter activity, but endogenous p53 is not able to regulate MMP-9 expression in first trimester CTB cells. Inactivation of p53 through mutation is the most common trait in cancer. By loosing its onco-suppressive activity, p53 becomes oncogenic in almost all malignant tumors (Soussi and Lozano, 2005). Although p53 is not mutated in the human placenta, it has become functionally incompetent. Understanding why and how p53 is functionally incompetent in CTB might well be the key to understanding trophoblast invasion.

Downregulation of EMMPRIN (BSG,CD147) by p53 leads to a decrease in the activity of MMP-9 and an inhibition of tumor cell invasion. Upregulation of EMMPRIN seen in many cancers can be attributed to, at least in part, to the dysfunction of p53 and thus provides new evidence for the roles of p53 in tumor development and progression. Epithelial derived MMP-9 exhibits a novel defensive role of tumor suppressor in colitis associated cancer by activating MMP9-Notch1-ARF-p53 axis. MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. 

The inter-activity of p53, cNK and MMP-9 are complexed, but this novel research may lead to the mechanisms by which arterial remodeling occurs after release of angiogenic factors from ECM. If that shares characteristics of NK invasion into developing tumor micro environment's a new therapeutic approach may arise.

 

Chemo vs. Mecho

Data strongly suggests interaction between plasma membrane and submembrane at the endothelial surface controls the inflammatory response. 

A meta-analysis from six studies of global gene expression profiles of Blood Pressure (BP) and hypertension was performed in 7017 individuals. 34 genes were differentially expressed. Of these, 6 genes were linked including MYADM, which was the only gene, of 34 discovered across diastolic, systolic BP and hypertension. Knockdown of MYADM (19q13), a component of endothelial surface rafts induced an inflammatory phenotype altering barrier function through the increase of the adhesion receptor ICAM-1 (19p13). This is mediated by MYADM activation of ERM actin cytoskeleton proteins. 

Mechanical forces, without a definitive direction e.g., disturbed flow and relatively undirected stretch at branch points and other complex regions cause sustained molecular signaling of pro-inflammatory and proliferative pathways that include mechanical stretch tied to p53. 

ERM proteins also facilitate Sphingosine-1-phosphate (S1P) dependent egress for T-cells to migrate from lymphoid organs. Their directional migration, by blebbing is contained at the T-cell’s leading edge. This fundamentally different mode of migration is characterized by intracellular pressurization. Of the five S1P receptors S1P2 (19p13) is critical in the immune, nervous, metabolic, cardiovascular, musculoskeletal, and renal systems. Results suggest that the ratio between S1P1 and S1P2 (19p13) governs the migratory behavior of different T cell subsets. 

Human NK cells express S1P1 mRNA. Activation with IL-2 increases S1P1, promotes S1P4 (19p13) and S1P5 (19p13) but not S1P2 (19p13) expression. Unlike S1P1, S1P2 (19p13) signals through several different G-alpha subunits, Gi, G12/13, and Gq. S1P5 (19p13) is also expressed in human and mouse NK cells and was required for mobilization to inflamed organs. S1P5-deficient mice had aberrant NK cell homing during steady-state conditions. NK cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor. 

Virus-infected mast cells selectively recruit NK cells and positively modulate their functions through mechanisms dependent on soluble mediators, such as interferons. Skin mast cells protect mice against vaccinia virus by triggering mast cell receptor S1P2 (19p13) and releasing antimicrobial peptides. S1P2 (19p13),  a negative regulator of platelet derived growth factor (PDGF) induced migration and proliferation as well as SphK1 expression. 

S1P inhibits macropinocytosis (internalizing extracellular materials) and phosphorylation of Akt via S1P2 (19p13) stimulation resulting in diminished antigen capture.

S1P1, S1P2 (19p13) and S1P3 receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development. S1P2 (19p13)  and S1P3 are involved in regulation of endothelial barrier function, fibrosis, and vasoconstriction. 

Adipogenic differentiation is inhibited by S1P2 (19p13) as mediated by C/EBPα and PPARγ, which induces PEPCK, a more recent gene of interest in cancer that acts at the junction between glycolysis and the Krebs cycle.

Mecho or chemo, chicken or egg, what first?

Systolic Blood Pressure and Innate Immunity vs. the Cancer Brain

Participants with a valid heart disease phenotype (atherosclerosis) were identified in a MESA blood pressure analysis conducted over 10 years. The valid group varied from 770 to 1113 patients from whom further blood analysis queried a primary and exploratory hypothesis of immune cell subsets. Four statistically significant innate cell subsets were discovered to be associated with Systolic blood pressure (SBP); Natural Killer (NK) cells, gamma delta T cells and classical monocytes.

Separately, an analysis of 7017 individuals from 6 international studies of gene expression signatures for SBP, diastolic blood pressure (DBP) and hypertension (HTN) found 7717 genes of which 34 were most differentialy expressed. Enrichment analysis for the systolic and diastolic gene group's associated to NK cell mediated cytotoxicity and 13 other pathways including antigen processing and inflammatory response, pointing strongly to innate and adaptive immunity. MYADM was the only gene identified for all groups SBP, DBP and HTN.

MYADM controls endothelial barrier function through ezrin, radixin, and moesin (ERM)-dependent regulation of ICAM-1 expression. ERM expression is required for ICAM-1 expression in response to MYADM suppression or TNF-α. ICAM-1 is a paradigmatic adhesion receptor that regulates leukocyte adhesion together with integrin LFA-1. This connection between endothelial membrane and cortical actin cytoskeleton appears to modulate the inflammatory response at the blood tissue barrier. 

Pressure overload activates the sympathetic nervous system (SNS) and up-regulates p53 expression in the cardiac endothelium and in bone marrow (BM) cells. Increased p53 expression promotes endothelial-leukocyte cell adhesion and initiates inflammation in cardiac tissue, which exacerbates systolic dysfunction. SNS activates, at least by significant increase of circulating norepinephrine (NE), which up-regulates p53 expressions, while forced expression of p53 increased ICAM-1 expression. 

On endothelial cells SNS is mediated via catecholamine-β2-adrenergic signaling, which up-regulates the production of reactive oxygen species (ROS), activates p53 and induces cellular senescence. Immune cells, including macrophages, monocytes, NK cells, B and T cells express the β2-adrenergic receptor and catecholamine. During pressure overload, NE cultured macrophages up-regulated p53 expression, whereas introduction of p53 increased Itgal (LFA-1) expression (which binds ICAM-1). Treatment with NE increased ROS, which was attenuated after inhibition of β2- adrenergic signaling in macrophages. Endothelial cell–macrophage interaction via NE-ROS-p53 signaling induces up-regulation of adhesion molecules, thus contributing to cardiac inflammation and systolic dysfunction.

During hypertension the vascular endothelium activates monocytes, in part through ROS by a loss of nitric oxide (NO) signaling, increased release of IL-6, hydrogen peroxide and a parallel increase in STAT activation in adjacent monocytes. NO inhibits formation of intermediate monocytes and STAT3 activation. Humans with hypertension have increased intermediate and non-classical monocytes and  intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells, and macrophages with activated STAT3.

A senescence-associated secretory phenotype (SASP) was induced in epithelial cells after DNA damage of sufficient magnitude. In premalignant epithelial cells SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy by a paracrine mechanism that largely depended interleukin (IL)-6 and IL-8. Strikingly, loss of p53 and gain of oncogenic RAS exacerbated the pro-malignant activities. This suggests a cell-non-autonomous mechanism by which p53 can restrain and oncogenic RAS can promote the development of age-related cancer by altering the tissue microenvironment. Oncogenic signaling pathways inhibit the p53 gene transcription rate through a mechanism involving Stat3, which binds to the p53 promoter in vitro and in vivo. Blocking Stat3 in cancer cells up-regulates expression of p53, leading to p53-mediated tumor cell apoptosis. 

Induced stretch or stretch from pressure overload may engage a non-autonomous, p53 centric micro-mechanical mechanism that escalates or deescalates innate responses against cells functioning outside the mechanical ranges that macrophages or NK cells permit. Thus, the neuro-immune extension through SNS signaling, may begin with circulating blood pressure or stretch promoted through inflammation. 

p53 vasoregulation and NK cell depletion in SARS-CoV2

p53 has earned first prize in the academic stakes. It is also the most mutated gene in cancer and elephant's have 20 copies, which probably explains their surprisingly low rate of cancer. Its associations to innate immunity, particularly Natural Killer (NK) cells through the mechanics of vasoconstriction-dilation have become a point of interest in COVID19 patients.

Remarkably COVID19 has inspired the global scientific community to focus a significant portion of its aggregate research toward the impact of  SARS-CoV2 (CoV2). For the first time in history global research is singularly focused because a large number of other protein's and gene's are affected by CoV2 binding Ace2. The Ace2 receptor is important in systems of vasoconstriction-dilation and has wide ranging impact.

CoV2 binding Ace2 reduces its availability to convert Angiotensin1 to Angiotensin 1-7 (Ang1-7) or Angiotensin 1-9 (Ang1-9), which primarily interact via MAS and Angiotensin2 Receptor (AT2R) respectively. These have been linked to signaling and stretch caused by vasoconstriction-dilation, mitochondrial dysfunction, mitochondrial fission as well as cardiac and vascular remodeling.

Ang1-7 and Ang1-9 interactions with MAS or AT2R cell surface receptors have been linked to signaling events that drive p53 binding DNA and transcription. Myocyte stretching activates p53 and p53-dependent genes, leading to the formation of Angiotensin II (Ang II) and apoptosis. AngII, stimulates phosphorylation of p53 (on serine 15) and CREB (on serine 133) and signaling converges on the p53-CRE enhancer to stimulate Bradykinin receptor 2 (BK2) gene transcription. BK2 is a key element in the p53 related kallikrein-kinin system (KKS) of vasodilation that counters the Renin-Angiotensin-Aldosterone-System (RAAS) of vasoconstriction. 

Aldosterone was shown to induce mitochondrial dysfunction and podocyte injury mediated by p53/Drp1-dependent mitochondrial fission. In neuronal cells p53 dependent declines in Drp1 and parkin contribute to altered mitochondrial morphology and cell death. Parkin, via Pink1 activity binds depolarized mitochondria to induce autophagy of mitochondria. Mutations in both Drp1 and Pink1 were fatal in Drosophila models. These events also implicate a direct functional link to chronic inflammation in ageing between p53 and expression levels of ICAM1 on endothelial and NK cells required to bind targets. The p53 mediated negative regulation of autophagy is Pink1 dependent and experiments have shown that mitochondrial antigens, recognized by NK cells presented on MHC's are Pink1 and parkin dependent. 

Severe COVID-19 patients have highly elevated Bradykinin and AngII, perhaps an indication of elevated p53 trends that have been discovered in these patients. Under normal circumstances, on endothelial cells Bradykinin would act as a potent vasodilator via its BK2 receptor. However, since Ang (1-7) potentiates Bradykinin action on BK2 receptors its near absence may reduce KKS vasodilation. On the other hand RAAS, also via p53 and elevated AngII primarily interacts with AT1R to promote vasoconstriction.  

NK cells through their Renin Angiotensin System may counter-regulate target cells in response. However, in COVID19 patients depletion of NK cells, invasion of Neutrophils and endothelial cell damage, in part through elevated p53 autophagy and apoptosis is the overwhelming nasty work of CoV2 against the backdrop of dysregulated blood pressure in tissue.

 

 

Impotent Natural Killers by Cancer Stem Cells and Ageing

Cancer stem cells have been found, through various mechanisms to alter the sentinel function and innate, immune surveillance of Natural Killer cells (NK). In senescent cells that have stopped cell division, including in cancer stem cell niches and NK induced vascular remodeling (as found in the developing placenta) NK's sentinel vigilance is also reduced.

Senescence-associated mitochondrial dysfunction, a significant trigger of multiple dimensions of the senescent phenotype is caused by disruption of normal mitochondrial autophagy (mitophagy). Mitophagy increases with aging and this age-dependent rise is abrogated by PINK1 or parkin deficiency. Deletion of a p53 response element on PINK1 promoter impacts p53-mediated PINK1 transcriptional repression. This p53-mediated negative regulation of autophagy has been found to be PINK1-dependent and constitutes a p53-PINK1 loop in nucleus and cytoplasm.

Further, mitophagy controls the activities of tumor suppressor p53 to regulate, at least hepatic cancer stem cells via Nanog. Prostate cancer cells escape NK attack by Nanog down-regulating ICAM1 (LFA1), to which NK would normally bind its target. In lung cancer NK have been found to limit the efficient clearance of senescent tumor cells from the mouse lung after p53 restoration. This indicated p53 may promote conditions for cellular survival and NK induced vascular remodeling or angiogenesis, necessary for the growth of tumors.

When under stress and inner mitochondrial membrane pressure gradient moves toward depolarization, Pink1 slots into the membrane, binds and phosphorylates p53 at Serine 392 (p53s392) and aids phagophore formation to enhance mitophagy. Mitophagy traps cytoplasmic p53s392, which reduces its transport to the nucleus where it would otherwise disrupt transcription of Nanog. (As illustrated below). 

Activated p53s392 nucleoside concentrations are effected by mitophagy

On the other hand, the sentinel function of NK may be subject to this PINK1 mediated mitochondrial switch. In prostate cancer cells Nanog promoted ICAM1 transcription required for NK binding target and cell killing. In prostate cancer cells Nanog over-expression restricts ICAM1, which promotes tumor formation. (As illustrated below). Investigating further, the direct functional link between p53 and ICAM-1 (CD54) in senescence and age-related disorders appears to be deeply integrated in mitophagy, senescence and immunity.

Nanog over-expression appears to be deterministic 

In stem cells where normal expression of Nanog transcribes ICAM1 and cancer stem cells where over-expression of Nanog restricts ICAM1, the variable PINK1-p53 switch may represent a "canary" that signals the state of  mitochondrial health to sentinel NK. However in some cancer cells where normal mitophagy is impaired and Nanog expression is restricted by p53s392, other p53 isoforms may directly promote the transcription of ICAM1.

In  two manipulation experiments using five different fibroblast cell lines that accelerated development of senescent associated secretory phenotypes a striking result was observed: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated pro-malignant paracrine signaling activities. Experiments show that PINK1 and Parkin, which are regulated by p53 specifically regulate mitochondrial antigen presentation of both MHC classes.

So, the question is whether the p53-PINK1 mitochondrial switch acts as cell-health "canary" for sentinel NK, where its inherent variables and regulatory loop may be fertile ground for the challenges of developing cancers?