Endothelial Research Articles

Unfolded protein response modulates the effects of GHRH antagonists in experimental models of in vivo and in vitro lung injury

ABSTRACT The development of efficient targeted therapies to ameliorate endothelial disorders is of the utmost need, as evident by the devastating outcomes of the recent pandemic. Recent findings suggest that unfolded protein response (UPR) modulates barrier function. In the current study, we reveal that the aforementioned highly conservative mechanism is involved in the protective effects of growth hormone-releasing hormone antagonists (GHRHAnt) in lung injury, both in vivo and in vitro. In bovine pulmonary artery endothelial cells, UPR suppression counteracted the protective effects of GHRHAnt in lipopolysaccharide (LPS)–induced endothelial hyperpermeability. In mouse lungs, UPR activation enhanced the beneficial effects of GHRHAnt against LPS-induced acute lung injury. Our observations – which are focused on lung endothelial cells and tissues – enhance our knowledge on the mechanisms mediating the barrier function and contribute to the development of novel therapies toward sepsis, direct and indirect lung injury. The effects of UPR modulation on the effects of GHRHAnt in other tissues are unknown, and they are the subject of future investigations.

The role of the inflammatory protein Pentraxin 3 as a biomarker in patients with ischemic heart disease

Atherosclerosis is a generalised process with a long latent period. Its manifestations in the context of ischemic heart disease are acute coronary syndrome and chronic coronary syndrome. Vascular inflammation is an integral part of the development and complications of atherosclerosis. Although primary and secondary prevention of ischemic heart disease has received much attention in clinical practice, the role of vascular inflammation remains incompletely understood and studied. Pentraxin 3 is an inflammatory biomarker that has the potential to be used for diagnostic and prognostic purposes in cardiology due to its local secretion from the vascular endothelium with peak values in the setting of acute coronary syndrome within 6-8 hours from symptom onset. The aim of this review is to examine the available evidence in the literature for its use in patients with ischemic heart disease.

Endothelial FUNDC1 Deficiency Drives Pulmonary Hypertension.

Pulmonary hypertension (PH) is associated with endothelial dysfunction. However, the cause of endothelial dysfunction and its impact on PH remain incompletely understood. We aimed to investigate whether the hypoxia-inducible FUNDC1 (FUN14 domain-containing 1)-dependent mitophagy pathway underlies PH pathogenesis and progression. We first analyzed FUNDC1 protein levels in lung samples from patients with PH and animal models. Using rodent PH models induced by HySu (hypoxia+SU5416) or chronic hypoxia, we further investigated PH pathogenesis and development in response to global and cell-type-specific Fundc1 loss/gain-of-function. We also investigated the spontaneous PH in mice with inducible loss of endothelial Fundc1. In addition, histological, metabolic, and transcriptomic studies were performed to delineate molecular mechanisms. Finally, findings were validated in vivo by compound deficiency of HIF2α (hypoxia-inducible factor 2α; Epas1) and pharmacological intervention. FUNDC1 protein levels were reduced in PH lung vessels from clinical subjects and animal models. Global Fundc1 deficiency exacerbated PH, while its overexpression is protective. The effect of FUNDC1 was mediated by endothelial cells rather than smooth muscle cells. Further, inducible loss of endothelial Fundc1 in postnatal mice was sufficient to cause PH spontaneously, whereas augmenting endothelial Fundc1 protected against PH before and after the onset of disease. Mechanistically, Fundc1 deficiency impaired basal mitophagy in endothelial cells, leading to the accumulation of dysfunctional mitochondria, metabolic reprogramming toward aerobic glycolysis, pseudohypoxia, and senescence, likely via a mtROS-HIF2α signaling pathway. Subsequently, Fundc1-deficient endothelial cells increased IGFBP2 (insulin-like growth factor-binding protein 2) secretion that drove pulmonary arterial remodeling to instigate PH. Finally, proof-of-principle in vivo studies showed significant efficacy on PH amelioration by targeting endothelial mitophagy, pseudohypoxia, senescence, or IGFBP2. Collectively, we show that FUNDC1-mediated basal mitophagy is critical for endothelial homeostasis, and its disruption instigates PH pathogenesis. Given that similar changes in FUNDC1 and IGFBP2 were observed in PH patients, our findings are of significant clinical relevance and provide novel therapeutic strategies for PH.

Endothelial Cell Senescence Effect on the Blood-Brain Barrier in Stroke and Cognitive Impairment.

Age is an important risk factor of stroke, cognitive decline, and dementia. Senescent endothelial cells (ECs) accumulate with advancing age through exposure to cellular stress, such as that exerted by hypertension and diabetes. These senescent ECs have altered characteristics, such as altered tight junction proteins, use of a more indiscriminate transcellular transport system, increased inflammation, and increased immune cell interactions. ECs are the main component of the blood-brain barrier (BBB), separating the brain from systemic circulation. As senescent ECs accumulate in the BBB, their altered functioning results in the disruption of the barrier. They have inadequate barrier-forming properties, disrupted extracellular matrix, and increased transcytosis, resulting in an overly permeable barrier. This disruption of the BBB can have important effects in stroke and cognitive impairment, as presented in this review. Besides increasing the permeability of the BBB, senescent ECs can also impair angiogenesis and vascular remodeling, which in ischemic stroke may increase risk of hemorrhagic transformation and worsen outcomes. Senescent ECs may also contribute to microvascular dysfunction, with disruption of cerebral perfusion and autoregulation. These may contribute to vascular cognitive impairment along with increased permeability. With an aging population, there is growing interest in targeting senescence. Several ongoing trials have been evaluating whether senolytics can slow aging, improve vascular health, and reduce the risk of stroke and cognitive decline.

Corneal Endothelial Cell Morphology in Eyes With Down Syndrome.

To explore the morphological characteristics of corneal endothelial cells in eyes with Down syndrome. We conducted a comparative analysis of corneal endothelial cell density, coefficient of variation, hexagonal cell appearance rate, and corneal thickness using specular microscopy. Our study included 21 eyes of 11 patients with Down syndrome and 37 eyes of 19 control subjects. In all eyes with Down syndrome, corneal endothelial cell density exceeded 2000 cells/mm2, and no abnormalities in cell morphology or significant differences from the control group were observed across all parameters. The structure and function of corneal endothelial cells in eyes with Down syndrome were within the normal range, even in the presence of chromosomal abnormalities of 21-trisomy in somatic cells.

Anxio-d epressive phenotype and impaired memory in mice with a conditional knockout of brain-derived neurotrophic factor in endothelial cells.

The present study investigated the role of endothelial BDNF in cognition. Male adult mice with a selective knockout of BDNF in endothelial cells (BDNFECKO) and their wild-type littermates (WT) were subjected to tests for detection of anxiety- and depression-like behaviors and impaired recognition memory. Neuronal activity and synaptogenesis were assessed from hippocampal levels of c-fos and synaptophysin, respectively, and cerebral capillary density from forebrain levels of CD31. BDNF/TrkB (tropomyosin-related kinase type B) receptor signaling was investigated through hippocampal levels of BDNF and activated TrkB receptors coupled with their immunolabelling by neurons and endothelial cells from both cerebrovascular fractions enriched in capillaries and hippocampal arterioles. Endothelial nitric oxide (NO) production was assessed from expression of endothelial NO synthase phosphorylated at serine 1177. BDNFECKO mice exhibited anxio-depressive phenotype, impaired memory and reduced synaptogenesis. Neither neuronal activity, neuronal BDNF/TrkB signaling nor capillary density differed between BDNFECKO and WT mice. However, endothelial-activated TrkB receptors as well as endothelial NO production and hippocampal BDNF levels were lower in BDNFECKO than WT mice. We conclude that endothelial BDNF is involved in cognition through mechanisms independent of neuronal BDNF/TrkB signaling and that endothelial NO might be a driver of the pro-cognitive effect of endothelial BDNF.

In vitro Examination of Piezo1-TRPV4 Dynamics: Implications for Placental Endothelial Function in Normal and Preeclamptic Pregnancies.

Mechanosensation is essential for endothelial cell (EC) function, which is compromised in early-onset preeclampsia (EPE), impacting offspring health. The ion channels Piezo-type mechanosensitive ion channel component 1 (Piezo1) and Transient receptor potential cation channel subfamily V member 4 (TRPV4) are co-regulated mechanosensors in ECs. Current evidence suggests that both channels could mediate aberrant placental endothelial function in EPE. Using isolated feto-placental ECs (fpECs) from early control (EC) and EPE pregnancies, we show functional co-expression of both channels and that Ca2+ influx and membrane depolarization in response to chemical channel activation is reduced in EPE fpECs. Downstream of channel activation, Piezo1 alone can induce phosphorylation of endothelial nitric oxide synthase (eNOS) in fpECs, while combined activation of Piezo1 and TRPV4 only affects eNOS phosphorylation in EPE fpECs. Additionally, combined activation reduces the barrier integrity of fpECs, also with a stronger effect on EPE fpECs. This implies altered Piezo1-TRPV4 co-regulation in EPE. Mechanistically, we suggest this to be driven by changes in the arachidonic acid metabolism in EPE fpECs as identified by RNA-Seq. Targeting of Piezo1 and TRPV4 might hold potential for EPE treatment options in the future.

Deciphering hematopoietic stem cell development: key signaling pathways and mechanisms

Most blood cells derive from hematopoietic stem cells (HSCs), originating from endothelial cells. The induction of HSCs from endothelial cells occurs during mid-gestation, and research has revealed multiple steps in this induction process. Hemogenic endothelial cells emerge within the endothelium, transition to hematopoietic cells (pre-HSCs), and subsequently mature into functional HSCs. Reports indicate transcription factors and external signals are involved in these processes. In this review, we discuss the timing and role of these transcription factors and summarize the external signals that have demonstrated efficacy in an in vitro culture. A precise understanding of the signals at each step is expected to advance the development of methods for inducing HSCs from pluripotent stem cells.

Highly Up-Regulation of Fas Ligand Gene Expression after Increasing in Oxidized Low-Density Lipoprotein

Oxidized low-density lipoprotein (ox-LDL) shows many harmful effects such as induction of apoptosis on function of endothelial cell (EC). Fas ligand (FASL) induces apoptosis in divergent pathological conditions. EC apoptosis which induced by ox-LDL is related with FASL. In this study, we aimed to determine how different ox-LDL levels affect FASL expression in ECs. We treated human umbilical vein endothelial cells (HUVECs) with two different concentration of ox-LDL. FASL gene expression in groups was detected by quantitative polymerase chain reaction (qPCR). We compared FASL gene expression level between the groups according to ∆∆Ct method. FASL gene expression was statistic significantly up-regulated in the group treated higher amounts of ox-LDL (P

Pyroptosis was suppressed by 20-hydroxyecdysone through Lin28b-mediated let-7d in vascular endothelial cells.

20-hydroxyecdysone (20E), a natural polyhydroxylated steroid, has exhibited anti-inflammatory effects across various diseases. This study investigates the potential connection between 20E's anti-inflammatory properties and the RNA-binding protein Lin28b, which is notably upregulated in TNF-α-stimulated endothelial cells. Herein, we discovered that 20E can selectively downregulate Lin28b expression without affecting its paralog Lin28a. Notably, 20E treatment could significantly attenuate pyroptosis, an inflammatory form of programmed cell death, as evidenced by reduced IL-1β and LDH release, and fewer propidium iodide (PI)-positive cells. Subsequent protein analysis revealed that 20E inhibited the enhanced expressions of key pyroptosis-associated proteins, GSDMD, GSDMD-N, and GSDME-N. Besides, this suppression of Lin28b and pyroptosis may be partially mediated through TNFR1. Additionally, 20E upregulated let-7 miRNA, particularly let-7d, a critical downstream target of Lin28b. To elucidate the role of Lin28b in 20E-mediated pyroptosis attenuation, we performed Lin28b overexpression and silencing experiments. Overexpressing Lin28b negated 20E's inhibition of LDH release and PI-related fluorescence, exacerbating GSDMD and GSDME cleavage. Conversely, Lin28b knockdown augmented the suppressive effect of 20E on pyroptosis, which was reversed by a let-7d inhibitor. Co-transfection with let-7d mimic and Lin28b plasmid demonstrated let-7d's role in mitigating pyroptosis aggravated by Lin28b overexpression. Overall, this study demonstrates that 20E may mitigate GSDMD and GSDME-mediated pyroptosis in HUVECs through the Lin28b/let-7d-dependent signaling pathway. These results highlight the potential of 20E as a promising inhibitor of pyroptosis, offering new insights into its anti-inflammatory mechanisms.

Dynamic behavior and lineage plasticity of the pulmonary venous endothelium.

Repair of the pulmonary vascular bed and the origin of new vasculature remain underexplored despite the critical necessity to meet oxygen demands after injury. Given their critical role in angiogenesis in other settings, we investigated the role of venous endothelial cells in endothelial regeneration after adult lung injury. Here we identified Slc6a2 as a marker of pulmonary venous endothelial cells and generated a venous-specific, inducible Cre mouse line. We observed that venous endothelial cells proliferate into the adjacent capillary bed upon influenza injury and hyperoxia injury. Imaging analysis demonstrated that venous endothelial cells proliferate and differentiate into general capillary and aerocyte capillary endothelial cells after infection, thus contributing to repair of the capillary plexus vital for gas exchange. Our studies thus establish that venous endothelial cells exhibit demonstrable progenitor capacity upon respiratory viral injury and sterile injury, contributing to repair of the alveolar capillary bed responsible for pulmonary function.

Pharmacologically inducing regenerative cardiac cells by small molecule drugs

Adult mammals, unlike some lower organisms, lack the ability to regenerate damaged hearts through cardiomyocytes (CMs) dedifferentiation into cells with regenerative capacity. Developing conditions to induce such naturally unavailable cells with potential to proliferate and differentiate into CMs, that is, regenerative cardiac cells (RCCs), in mammals will provide new insights and tools for heart regeneration research. In this study, we demonstrate that a two-compound combination, CHIR99021 and A-485 (2C), effectively induces RCCs from human embryonic stem cell-derived TNNT2+ CMs in vitro, as evidenced by lineage tracing experiments. Functional analysis shows that these RCCs express a broad spectrum of cardiogenesis genes and have the potential to differentiate into functional CMs, endothelial cells, and smooth muscle cells. Importantly, similar results were observed in neonatal rat CMs both in vitro and in vivo. Remarkably, administering 2C in adult mouse hearts significantly enhances survival and improves heart function post-myocardial infarction. Mechanistically, CHIR99021 is crucial for the transcriptional and epigenetic activation of genes essential for RCC development, while A-485 primarily suppresses H3K27Ac and particularly H3K9Ac in CMs. Their synergistic effect enhances these modifications on RCC genes, facilitating the transition from CMs to RCCs. Therefore, our findings demonstrate the feasibility and reveal the mechanisms of pharmacological induction of RCCs from endogenous CMs, which could offer a promising regenerative strategy to repair injured hearts.

Pharmacologically inducing regenerative cardiac cells by small molecule drugs.

Adult mammals, unlike some lower organisms, lack the ability to regenerate damaged hearts through cardiomyocytes (CMs) dedifferentiation into cells with regenerative capacity. Developing conditions to induce such naturally unavailable cells with potential to proliferate and differentiate into CMs, that is, regenerative cardiac cells (RCCs), in mammals will provide new insights and tools for heart regeneration research. In this study, we demonstrate that a two-compound combination, CHIR99021 and A-485 (2C), effectively induces RCCs from human embryonic stem cell-derived TNNT2+ CMs in vitro, as evidenced by lineage tracing experiments. Functional analysis shows that these RCCs express a broad spectrum of cardiogenesis genes and have the potential to differentiate into functional CMs, endothelial cells, and smooth muscle cells. Importantly, similar results were observed in neonatal rat CMs both in vitro and in vivo. Remarkably, administering 2C in adult mouse hearts significantly enhances survival and improves heart function post-myocardial infarction. Mechanistically, CHIR99021 is crucial for the transcriptional and epigenetic activation of genes essential for RCC development, while A-485 primarily suppresses H3K27Ac and particularly H3K9Ac in CMs. Their synergistic effect enhances these modifications on RCC genes, facilitating the transition from CMs to RCCs. Therefore, our findings demonstrate the feasibility and reveal the mechanisms of pharmacological induction of RCCs from endogenous CMs, which could offer a promising regenerative strategy to repair injured hearts.

Cytochrome P450-derived Epoxyeicosatrienoic Acid, the Regulation of Cardiovascular-related Diseases, and the Implication for Pulmonary Hypertension.

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid (AA) into biologically active epoxyeicosatrienoic acids (EETs), forming a pivotal metabolic pathway (AA-CYP-EETs-soluble epoxide hydrolase-dihydroxyeicosatrienoic acids) implicated in the progression of various disorders. Inflammation is a key contributor to the onset and progression of numerous systemic diseases, and EETs play a significant role in mitigating inflammation. Extensive research highlights the cardiovascular protective effects of EETs, which include vasodilation, anti-hypertensive, and anti-atherosclerotic properties. Interestingly, the relatively less-explored third metabolic pathway of AA exhibits both pro-proliferative and anti-apoptotic effects in endothelial cells and smooth muscle cells. Recent studies have shown elevated levels of EETs catalyzed by CYP epoxygenases in human tumors, promoting tumor progression and metastasis-phenomena closely related to the disease progression in pulmonary hypertension (PH). This review explores the current understanding of the regulatory functions of CYP-derived EETs in cardiovascular diseases and seeks to elucidate their potential implications in PH. Ultimately, understanding the multifaceted roles of EETs may help identify novel therapeutic targets for both cardiovascular diseases and PH.

Decoding Angiocrine Signaling: Endothelial Cells as Drivers of Organ Regeneration and Homeostasis.

Decoding Angiocrine Signaling: Endothelial Cells as Drivers of Organ Regeneration and Homeostasis.

ADAM10-mediated β-klotho degradation: a key to FGF21 resistance in senescent vascular endothelial cells.

Fibroblast growth factor 21 (FGF21) is a key regulator of metabolism and cardiovascular health. However, its upregulation in aging and age-related disorders suggests the presence of FGF21 resistance. This study aimed to elucidate the mechanisms underlying senescence-associated FGF21 resistance in human umbilical vein endothelial cells (HUVECs) and to explore potential therapeutic interventions. Transcriptomic analysis revealed a significant reduction in the number of FGF21-regulated genes in senescent HUVECs compared to young cells, indicating the onset of FGF21 resistance. In young HUVECs, FGF21 inhibited inflammatory cytokines and upregulated mitochondrial ribosomal protein genes. Conversely, senescent HUVECs showed a downregulation of β-klotho, an essential co-receptor for FGF21, at the cell membrane, concomitant with an upregulation of ADAM10, a protease involved in β-klotho degradation. These changes were also observed in aged mouse aortas. ADAM10 was shown to bind directly to β-klotho and promote its degradation, contributing to FGF21 resistance. Inhibition of ADAM10 or overexpression of β-klotho restored FGF21 responsiveness in senescent HUVECs. Moreover, pharmacologically high concentrations of FGF21 were effective in overcoming resistance and restoring its regulatory effects on senescent cells. These findings suggest that ADAM10-mediated degradation of β-klotho is a central mechanism in the development of FGF21 resistance, and that targeting the ADAM10/β-klotho axis could represent a novel therapeutic approach to restore FGF21 sensitivity. Pharmacological administration of FGF21 may hold promise in treating vascular aging and its associated cardiovascular pathologies.

Relaxin-2 improves type I diabetes mellitus-induced erectile dysfunction in rats by protecting cavernous endothelial and smooth muscle function, and inhibiting penile fibrosis and apoptosis.

Diabetes mellitus-induced erectile dysfunction (DMED) responds poorly to first-line treatments, necessitating the development of new therapeutic strategies. Relaxin-2 (RLX-2) plays a crucial role in protecting vascular endothelium, vasodilatation, and antifibrosis in various diseases. However, its effects and mechanisms on DMED remain unclear. To investigate the effects and mechanisms of RLX-2 on DMED rats in vivo and vitro. For in vivo research, 30 Sprague-Dawley rats were allocated into three groups: control, DMED, and DMED + RLX-2. The induction of DMED in the rats was achieved through intraperitoneal administration of streptozotocin, with confirmation of ED status being conducted via the apomorphine test. Rats in the DMED + RLX-2 group received continuous RLX-2 treatment by osmotic pump. Following a 4-week treatment period, assessment of erectile function was carried out using cavernous manometry, and samples of corpus cavernosum tissues were procured for subsequent analysis. For in vitro research, human cardiac microvascular endothelial cells (HCMECs) were allocated into three groups: control, high glucose (HG, 40mM), and HG + RLX-2. HCMECs were cultured for 6 days and treated with RLX-2 for 48h before collection for subsequent experiments. In DMED rats, RLX-2 treatment partially improved erectile function. We observed relatively normalized functions of endothelial and smooth muscle cells with decreased levels of apoptosis and fibrosis in the penis. In vitro experiments also demonstrated the antihyperglycemic effects of RLX-2. RLX-2 can protect endothelial and smooth muscle function, and inhibit aberrant apoptosis and fibrosis in the corpus cavernosum, thereby improving erectile function in DMED rats. This may provide a novel treatment for DMED.

Chronic inflammation and vascular cell plasticity in atherosclerosis.

Vascular smooth muscle cells, endothelial cells and macrophages undergo phenotypic conversions throughout atherosclerosis progression, both as a consequence of chronic inflammation and as subsequent drivers of it. The inflammatory hypothesis of atherosclerosis has been catapulted to the forefront of cardiovascular research as clinical trials have shown that anti-inflammatory therapy reduces adverse cardiovascular events. However, no current therapies have been specifically designed to target the phenotype of plaque cells. Fate mapping has revealed that plaque cells convert to detrimental and beneficial cell phenotypes during atherosclerosis, with cumulative evidence highlighting that vascular cell plasticity is intimately linked with plaque inflammation, ultimately impacting lesion stability. Here we review vascular cell plasticity during atherosclerosis in the context of the chronic inflammatory plaque microenvironment. We highlight the need to better understand how plaque cells behave during therapeutic intervention. We then propose modulating plaque cell phenotype as an unexplored therapeutic paradigm in the clinical setting.

P. gingivalis-Infected Macrophage Extracellular Vesicles Cause Adverse Pregnancy Outcomes.

Periodontitis is a chronic inflammatory disease triggered by oral bacterial infection, with the bacterium Porphyromonas gingivalis being a major causative agent. The association between periodontitis and various systemic diseases has been demonstrated. Recent research has also highlighted the relationship between the aggravation of maternal periodontitis and adverse pregnancy outcomes such as preterm birth and low birth weight. However, the molecular mechanisms underlying how factors from periodontitis influence pregnancy and fetal development remain unclear. Extracellular vesicles (EVs) are nano-sized spherical particles secreted into the tissue microenvironment by various types of cells. EVs have garnered interest in recent years due to their role in intercellular communication. In the present study, we investigated whether EVs derived from P. gingivalis-infected macrophages (Pg-inf EVs) reach the feto-placental unit and influence fetal development. Through a series of in vivo experiments in mice, we demonstrated that Pg-inf EVs translocated to the feto-placental unit and impaired fetal development in size and weight. Histological analysis revealed disoriented blood vessel alignment and impaired angiogenesis in the placentas of Pg-inf EV-injected groups, indicative of compromised placental function. Proteome analysis revealed a significant decrease in Vegfr1 expression in the placentas of the experimental group. Moreover, Pg-inf EVs reduced VEGFR1 expression in cultured human vascular endothelial cells, highlighting a potential molecular mechanism through which these EVs exert their effects on placental angiogenesis. This is the first study to reveal a novel pathway in which oral bacteria-infected macrophage EVs in maternal periodontitis affect pregnancy via the feto-placental unit.

Angiogenic effects of Type 2 diabetes on the dental pulp.

To investigate the influence of type 2 diabetes (T2D) and hyperglycaemia on blood vessels and angiogenic markers in the dental pulp. Extracted non-carious permanent molar teeth were collected from patients with well-controlled T2D (n = 10) and non-T2D (controls) (n = 10). The pulp was examined qualitatively using haematoxylin and eosin and Van Gieson stains. Immunohistochemistry (IHC) identified the primary receptor for VEGF, VEGFR2, and the endothelial cell marker CD34. Primary human dental pulp cell (hDPC) lines (n = 3) were established from tissue explants and cells were grown in media containing 5.5 mM D-glucose (control), 12.5 mM (prediabetes) and 25 mM (T2D) D-glucose under normoxic conditions for 24, 48 and 72 h. Assays for metabolic activity (PrestoBlue) and cell viability (Crystal Violet staining) assessed the hDPC response to hyperglycaemia. The expression of angiogenic genes VEGFA, KDR, FLT-1, ANGPT1, ANGPT2, TIE1 and TEK were analysed using quantitative real-time polymerase chain reaction. ELISAs were used to quantify the level of expressed protein for VEGFA, ANG1, ANG2, TIE1, and TIE2 in the media. Data analyses were performed using GraphPad Prism and anova tests at p < .05. Blood vessels in T2D samples had thicker walls and stained strongly for elastin and collagen compared with non-T2D samples. VEGFR2 protein was not seen in any T2D samples but consistently detected in healthy specimens. Culturing healthy cells in high glucose (25 mM) significantly reduced cell viability at 24 h compared to the control (p = .005) and 12.5 mM glucose (p = .001) but the metabolic activity was not greatly affected by glucose and time. VEGFA mRNA and VEGFA protein expression were detected in the hDPCs in the presence of hyperglycaemia over time; however, the primary receptor, VEGFR2/KDR, was not detected. Genes for the ANG1 and ANG2 and their receptors were expressed at all glucose concentrations but hyperglycaemia upregulated ANG2 mRNA. Proteins for all growth factors were detected in the media however proteins for TIE1 and TIE2 receptors were not. T2D and hyperglycaemia may impair the angiogenic response in the pulp similar to other body site. The scarcity of VEGFR2 and increased expression of ANG2 in response to hyperglycaemia suggests that VEGF and ANG-Tie1/Tie2 signalling may be compromised.