An Introduction to Primary Endothelial Cells

Endothelial cells form a one-cell thick walled layer called endothelium that lines all of our blood vessels. Depending on the tissue of origin, endothelial cells fulfill different functions. Endothelial cells derived from the kidney, will exhibit functions related to filtration while endothelial cells sourced from the brain maintain the blood-brain-barrier as a vital mechanism of protection against any harmful agents which may be circulating.

Learn more about endothelial cells and their functions and browse this page for a wealth of technical information including many white papers and selected citations. Don't miss our application area further down on this page and read how endothelial cells are being used to study complex processes related to wound healing, angiogenesis, inflammation, blood brain barriers, diabetes and other cardiovascular diseases.

If you are familiar with endothelial cell culture and are interested in purchasing Lonza's cells or media products, please find the quick links to our products below:

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Many of the endothelial processes are commonly studied in vitro using human umbilical vein endothelial cells (HUVEC), or large vessel and microvascular endothelial cells isolated from various body tissues. Lonza offers a comprehensive range of normal human primary endothelial cells and supporting media for cardiac, aortic, coronary iliac artery, pulmonary artery, cardiac microvascular, umbilical vein, dermal, and lung cells. In addition, we offer endothelial cells from donors diagnosed with diabetes type I or type II from various tissue origins.

Endothelial Cells

Lonza’s EGM^TM Endothelial Growth Media products have been optimized for large vessel and microvascular endothelial cells from various tissue origins. Our BulletKit^TM Media is formulated with additional growth factors and hormones to optimally support consistent growth of primary cells while maintaining the tissue-specific characteristics. Our archive of publications and supporting white papers show suitability of our endothelial media products for establishing complex co-culture models or developing advanced cell culture models.

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What are Endothelial Cells and their Function?

The endothelial cells form a one-cell thick walled layer called endothelium that lines all of our blood vessels such as arteries, arterioles, venules, veins and capillaries. Smooth muscle cells layer beneath the endothelial cells to form the blood vessel.

The largest blood vessels are arteries and veins, which have a thick, tough wall of connective tissue and many layers of smooth muscle cells¹. The wall is lined by an exceedingly thin single sheet of endothelial cells separated from the surrounding outer layers by a basal lamina.

The amounts of connective tissue and smooth muscle in the vessel wall vary according to the vessel's diameter and function, but the endothelial lining is always present. In the finest branches of the vascular tree—the capillaries and sinusoids—the walls consist of nothing but endothelial cells and a basal lamina, together with a few scattered—but functionally important pericytes.

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Endothelium is classified as continuous, fenestrated or discontinuous. Capillaries with a continuous endothelium are found in the lungs, muscle and central nervous system. Within continuous endothelium, the endothelial cells are joined by tight junctions,anchored to a continuous basal membrane and contain negatively charged complexes that regulate the passage of molecules out of tissue to the blood stream.

Fenestrated endothelium is characterized by the presence of perforations of 50 – 60nm (fenestrae), which make them more permeable than continuous endothelium. Fenestrated endothelium is usually found in kidney and villi of intestine. The pores expand and contract in response to stimuli which include hormones, neurotransmitters, nicotine and alcohol. Similar to continuous endothelium, fenestrated endothelium is secured to a continuous basal membrane. These blood vessels allow for passive transport across the endothelium, which makes them a key component in the filtration role, within the gastrointestinal tract and kidney glomeruli.

Discontinuous endothelium also contains pores however these are considerably larger than those of fenestrated endothelium and do not have a diaphragm. The basal membrane is discontinuous. This form of endothelium exists in the blood vessels of the spleen, liver and bone marrow and has greater permeability than continuous or fenestrated endothelium.

Functions of Endothelial Cells

The endothelium serves as a permeable barrier for the blood vessel and is involved in the regulation of blood flow. Within basic research, endothelial cells are pivotal to applications related to wound healing, angiogenesis, inflammatory processes, blood brain barriers, diabetes and other cardiovascular diseases. Since the circulatory system lines the entire body, endothelial research is tied to many diseases, which are top-funded research areas. For this reason, dysfunction of endothelial cells has implications in diabetes, pulmonary diseases, inflammatory diseases, cardiovascular diseases and immune diseases to name a few.

In addition, blocking angiogenesis has important applications in cancer. Cancerous tissue is as dependent on a blood supply as is normal tissue, and this has led to a surge of interest in endothelial cell biology. Studies are focused on understanding interactions between blood vessel formation and tumor expansion. Endothelial cells also have important applications in tissue engineering studies especially in the development of artificial vascular grafts as alternatives to donor-sourced grafts or supporting organ or tissue engineering with a vascular network. Endothelial cells have a remarkable capacity to adjust their number and arrangement to create an adaptable life-support system, extending by cell migration into almost every region of the body. If it were not for endothelial cells extending and remodeling the network of blood vessels, tissue growth and repair would be impossible.

Barrier Function

The endothelium acts as a barrier between the blood and the rest of the body tissue while being selectively permeable to certain chemicals and white blood cells to move across from blood to tissue or for waste and carbon-dioxide to move from tissue to blood. This property of endothelial cells is especially investigated in the blood-brain-barrier system. In certain neuro-degenerative diseases, it is difficult to develop drugs that can cross the endothelial barrier efficiently. Research is focused on better mimicking and understanding the functions of blood brain barrier systems to increase the efficacy of drug development.

Illustration of blood brain barrier model

Image courtesy of Pharmaceutics 2014, 6(4), 557-583; doi:10.3390/pharmaceutics6040557

Regulation of Blood Flow

Vasodilation and vasoconstriction, the widening and narrowing of blood vessels respectively, are essential processes to controlling blood flow. The endothelium responds to various vasoactive factors to maintain the vascular tone of arteries and veins and achieves this via the contraction or relaxation of the smooth muscle cells which underlie the basal membrane of these vessels. Nitric oxide, produced by many cells in the body, is a vasodilator. Norepinephrine, which functions as a hormone and a neurotransmitter, is a vasoconstrictor. Endothelial cells generate an anti-thrombotic surface that facilitates transit of plasma and cellular constituents throughout the vasculature and maintenance of homeostasis.

Wound Healing

Hemostasis, the process by which bleeding is stopped at the site of an injury while normal blood flow is maintained throughout the circulatory system, is one of the first stages of wound healing. The endothelium is pivotal to this, for example by controlling platelet adhesion and activation and by synthesizing the essential blood clotting protein Factor VIII. Endothelial cells are also involved in fibrinolysis, the dissolution of blood clots once the wound has healed. They can achieve this by secreting tissue-type plasminogen activator (t-PA).

Prevention of Thrombosis

Thrombosis is the abnormal formation of a blood clot within the vasculature which can obstruct blood flow. Although blood clotting is an essential stage of the wound healing process, it can be fatal if allowed to occur in an uncontrolled manner. The endothelium has various anti-thrombotic properties. These include the secretion of prostacyclin which inhibits platelet aggregation, and the storage and release of von Willebrand factor which binds to the clotting protein Factor VIII.

Angiogenesis

Angiogenesis, the formation of new blood vessels, occurs during early embryonic development and continues throughout the entire human lifespan. It is essential to the process of wound healing but has particularly been a subject of interest to researchers who study tumorigenesis. The proliferation and metastasis of cancer cells is dependent on an adequate supply of oxygen and nutrients. Tumor growth is supported by formation of new blood vessels that provide nutrients for these cells to expand. Many cancer therapeutic strategies have targeted tumor angiogenesis in hopes of limiting cancer progression.

The Inflammatory Response

Endothelial cells are key regulators of the inflammatory response since they form a crucial line of defense against infection. Dysregulation of the inflammatory response is implicated in a wide variety of diseases, and researchers have studied many different signaling pathways to advance understanding of the role of endothelial cells in conditions which include atherosclerosis, rheumatoid arthritis and inflammatory bowel diseases.

Endothelial Cells In Vitro

While endothelial cells show heterogeneity among different tissue origins in terms of genes they express, they are typically similar in morphology. Endothelial cells consist of a "cobblestone" morphology, stain positive for Factors VIII (an essential blood-clotting protein synthesized by endothelial cells) and take up acetylated low-density lipoprotein. HUVECs stain positive for CD-31.

Primary endothelial cell diversity revealed by global expression profiling of 53 different lots isolated from 14 distinct tissue origins. The data illustrates varying gene expression profile of endothelial cells based on different vessel sizes and origin of cells (veins or arteries). The differences were conserved over a few passages. Chi JT. et al. (2003) PNAS 100:10623-8. By permission of the author. Copyright 2003 National Academy of Sciences.

References

¹ Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Blood Vessels and Endothelial Cells.

Application – Blood Brain Barrier Models

The endothelium acts as a barrier between blood and rest of the body tissue. It is selectively permeable for certain chemicals and white blood cells to move across from blood to tissue or for waste and carbon-dioxide to move from tissue to blood.

The blood-brain barrier (BBB) is formed by microvascular endothelial cells, pericytes and astrocytes. It prevents the entry of most large hydrophilic molecules and many potentially harmful toxins from the blood into the brain. On the other hand, it also prevents the entry of many therapeutic agents into the brain making it difficult to develop drugs that can efficiently cross the blood-brain-barrier. Considerable efforts are made to better mimic and understand the functions of blood brain barrier systems to increase the efficacy of drug development.

The endothelium acts as a barrier between blood and rest of the body tissue. It is selectively permeable for certain chemicals and white blood cells to move across from blood to tissue or for waste and carbon-dioxide to move from tissue to blood. The blood-brain barrier (BBB) is formed by microvascular endothelial cells, pericytes and astrocytes. It prevents the entry of most large hydrophilic molecules and many potentially harmful toxins from the blood into the brain. On the other hand, it also prevents the entry of many therapeutic agents into the brain making it difficult to develop drugs that can efficiently cross the blood-brain-barrier. Considerable efforts are made to better mimic and understand the functions of blood brain barrier systems to increase the efficacy of drug development.

Image on the left:

A schematic sketch of blood vessels in the brain By Armin Kübelbeck, CC BY 3.0

Spampinato et al. studied the effects of bioactive lipid S1P and fingolimod (an immune drug) on the BBB models. In this figure, human brain microvascular endothelial cells were cultured in EGM^TM-2 SingleQuot Kits and immunostained for the cytoskeleton component F-actin. Fig a. shows a confluent monolayer characterized by fusiform morphology and strict cell-to-cell contacts. Fig b shows exposure to inflammatory cytokines, TNFα and IFNγ, causing changes in hBMVEC morphology as consequence of reduced cell viability. In the presence of S1P (Fig c) or fingolimod phosphate (Fig d), effects of inflammatory cytokines on hBMVEC morphology are lessened. (CC BY 4.0)

Selected References with Lonza’s EGM^TM-2 Endothelial Growth Medium for Developing Blood-Brain-Barrier Models with Brain Microvascular Endothelial Cells

HCMEC/D3 cell line is commonly used as an vitro model to develop the BBB models. Vu et al. demonstrates how HCMEC/D3 cell line can be grown in rich endothelial growth medium EGM^TM-2 on a collagen-coated porous membrane. The researchers were studying whether C. neoformans can cross the brain endothelium transcellularly without causing significant damage to the endothelial cells
Human Brain MicroVascular Endothelial Cells (hBMVEC), were grown in MCDB-131 media supplemented with EGM^TM-2 SingleQuots. Spampinato et al. examined the ability of S1P(a bioactive sphigolipid) and fingolimod (an immune modulatory drug) to act on the BBB

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Application – Angiogenesis

Angiogenesis is a term used to refer to “formation of new blood vessels”. Endothelial cells play a key function in maintaining homeostasis and formation of new blood vessels. “Angiogenesis involves the migration, growth, and differentiation of endothelial cells, which line the inside wall of blood vessels.” ¹

Angiogenesis has been found to have key applications in cancer research. Tumor is supported by formation of new blood vessels that provides oxygen nutrients for cancer cells to grow and invade other tissue. Current research is focused on understanding how natural and/or synthetic angiogenesis inhibitors, also known as antiangiogenic agents, can have implications on stopping or lessening tumor growth. The key role of angiogenesis in tumor development and cancer metastasis makes inhibition of angiogenesis an attractive strategy to target a number of cancer types (Timar et. al., 2001).

Human umbilical vein endothelial cells (HUVEC) are commonly used primary endothelial cell types for studying angiogenesis in vitro. VEGF (or VEGF-A “vascular endothelial growth factor”) and VEGFR-2 are key cytokines involved in stimulating angiogenesis. One of the easiest screening and target validation strategies for anti-angiogenic target identification involves knocking down targets in HUVECs and assessing subsequent effects on tube formation. The usage of small interfering RNA (siRNA) is one of the strategies to knock-down RNA, and thereby protein expression within cells. siRNA can be delivered within cells using either chemical transfection or electroporation-based strategies such as the one offered by the Lonza Nucleofector^TM Technology.

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Angiogenesis and Cancer

The properties and behavior of cancer cells and tumoroids are strongly influenced by the surrounding extracellular matrix. Therefore each meaningful oncology model should contain a representative extracellular matrix. Tumor progression is mediated by micro-environmental conditions that include cell-cell and cell-extracellular matrix (ECM) interactions. Tumor metastasis is influenced by the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumor microenvironment.

Learn more and read the full scientific article Engineering a vascularised 3D in vitro model of cancer progression.

Select References with Lonza’s HUVEC and EGM^TM-2 Medium for Angiogenesis Assays

Legeay et al , in their study, show Lonza’s HUVECs were cultured in EGM-2 Medium and exposed to DEET found in mosquito repellants. The team showed how DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth.
Wang et al demonstrates Cetuximab inhibits tumor angiogenesis in vitro. Pooled HUVECs from Lonza were cultured in EGM-2 medium and migration assay was studied in the presence of pretreated medium with Cetuximab and Cal27 cells

Wang et al demonstrates Cetuximab inhibits tumor angiogenesis in vitro. The research group performed an in vitro migration assay with Lonza’s pooled HUVECs to further confirm the function of cetuximab in angiogenesis in vitro. As shown in Fig. B, the findings exhibited that conditional medium (CM) significantly decreased HUVEC migration and tube formation after cetuximab pretreatment under both normoxic and hypoxic conditions when compared with the negative vehicle. (CC0 1.0)

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References

¹ https://www.cancer.gov/about-cancer/treatment/types/immunotherapy/angiogenesis-inhibitors-fact-sheet

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Application – Tissue Engineering

The first tissue-engineered vascular graft (TEVG) was implanted in a child over a decade ago. With cardiovascular diseases becoming more prevalent and the need the limited availability of healthy autologous vessels for bypass grafting procedures, there is an increased need to look for alternative sources to increase treatment efficiency. This has fueled the growth of in vitro vascular tissue engineering field as driven by clinical demand for improved vascular prostheses with performance and durability similar to an autologous blood vessel.”¹

Advancements are being made in vitro for testing new methods for vascular graft engineering ranging from seeding cells into decellularized naturally-derived scaffolds to using synthetic scaffolds and testing cell adherence. For such investigations, mimicking in vivo conditions as closely as possible becomes vital and hence primary cultures of endothelial and smooth muscle cells are utilized as ideal cell sources to advance research in this arena.

As an example, the attachment of Lonza HUVEC cells to modified graft surfaces, and their subsequent proliferation, was assessed. The rationale behind this work was centered around the potential for tissue engineered vascular grafts to act as biodegradable scaffold for cells to attach to, proliferate, and provide physiologic functionality. Such a scaffold would eliminate many of the problems associated with permanent synthetic grafts. The authors of the study investigated two endothelialization strategies – antibody immobilization or loading of vascular endothelial growth factor (VEGF). The modified graft surfaces demonstrated superior endothelial cell attachment in vivo, indicating the methodology to offer significant improvements over synthetic alternatives for tissue engineering (Melchiorri et. al)

In a separate study Bastijanic et. al utilized Lonza’s Human pulmonary artery endothelial cells (HPAECs ) and human coronary artery smooth muscle cells (HCASMCs ) and cultured them to 80–90% confluency in endothelial cell growth medium (EGM) or smooth muscle cell growth medium (SmGM), respectively. The cells were integrated individually within synthetic vascular scaffolds to test for cell adherence showing promising results.

Histological examination of the luminal side of the vascular graft

At pre-implantation, the vascular endothelial cells distribute to the entire area of the graft (data not shown). Conversely, after implantation, vWF, CD31 and CellTracker Red-positive endothelial cells are seen at the inner lumen of the vessel. Furthermore, the vascular endothelial cells cover the inner surface of the vessel more continuously on the fifth day than on the second.

Itoh, et al developed a novel method to create scaffold-free tubular tissue from multicellular spheroids (MCS) using a “Bio-3D printer”-based system. The research team made scaffold-free tubular tissues from MCSs composed of Lonza’s human umbilical vein endothelial cells, human aortic smooth muscle cells, and normal human dermal fibroblasts. The tubular tissues were cultured in a perfusion system and implanted into the abdominal aortas of F344 nude rats. The scaffold-free tubular tissues made of MCS using a Bio-3D printer underwent remodeling and endothelialization. (CC BY 4.0)

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Large vessel and microvascular endothelial cells and smooth muscle cells from normal and diabetic donors

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Application – Inflammatory Processes

Inflammation is body’s natural process of removing harmful stimuli and begin the healing process. Endothelial cells have been found to function as key modulators of the inflammatory processes. Consequently, dysfunction in endothelial cells can result in implications to inflammatory processes as well. Inflammation has also been linked to many disease areas including cardiovascular, pulmonary, neurological, autoimmune, diabetes to name a few. Therefore, primary endothelial cells become crucial components supporting research in many disease areas.

According to an article by NatureReviews, microvascular endothelial cells (HMVEC) regulate and are active participants in inflammatory processes at a site of inflammation. The properties of endothelial cells change from acute to chronic inflammation and from innate to adaptive immunity. The article concludes that since inflammatory processes and endothelial cells are correlated, many anti-inflammatory therapies influence the behavior of endothelial cells and vascular therapeutics influence inflammation.

Sickle Cell Disease – Current Treatment and New Therapeutical Approaches

Effects of nitric oxide on the vascular endothelium, inflammatory cells, and platelets. The image shows effects of NO impacting inflammatory cells and endothelial cells.

Select References with Lonza’s Primary Endothelial Cells as Regulators of Inflammatory Processes

Tellier, et al. stated that numerous studies have evidenced different consequences of cycling hypoxia versus chronic hypoxia on cancer cells as well as on endothelial cell behavior, altering angiogenesis, tumor growth, and metastasis. Endothelial cells, key regulators of tumor growth, differentially respond to changes in tumor microenvironment. The team hypothesized that endothelial cells could be a perceptive sensor of cycling hypoxia and inflammation simultaneously. Lonza’s HUVEC and EGM growth medium were used with colon cancer tissue to perform the study. Results showed that endothelial cells exposed to cycling hypoxia displayed an amplified inflammatory phenotype.
Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions. Dong, et al. sought to better understand the molecular mechanism of how cells respond to acidic tissue microenvironment. The team was investigating GPR4, a protein expressed by endothelial cells and also activated under acidic conditions. Several endothelial cell types from Lonza including human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (HMVEC-L) and human pulmonary artery endothelial cells (HPAEC) were utilized and grown in endothelial cell growth medium 2 (EGM-2), or EGM-2-MV medium. Gene expression of the acidosis response was compared with varying levels of GPR4. Acidosis activation of GPR4 demonstrated a broad inflammatory response with substantial increases in the expression of inflammatory genes such as chemokines, cytokines, adhesion molecules and NF-kB pathway genes. Suppression of this response by GPR4 inhibitors was considered to have significant therapeutic potential in pathological conditions including ischemia, sickle cell disease, tumor, metabolic diseases, renal diseases, and respiratory diseases

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Application – Diabetes and Cardiovascular Diseases

Endothelial dysfunction causes an imbalance between vasodilation and vasoconstriction. The term, endothelial dysfunction, has been used to refer to altered anticoagulant and anti-inflammatory properties of the endothelium, impaired modulation of vascular growth, and dysregulation of vascular remodeling1. Endothelial dysfunction is tied to an impairment of endothelium-dependent vasorelaxation caused by a loss of nitric oxide (NO) bioactivity in the vessel wall. This endothelial dysfunction has been implicated in many vascular diseases such as atherosclerosis, diabetes, hypertension and more. Impaired endothelium function in the coronary circulation has been found to have prognostic implications in that it predicts adverse cardiovascular events and long-term outcome¹.

“Substantial clinical and experimental evidence suggest that both diabetes and insulin resistance cause a combination of endothelial dysfunctions, which may diminish the anti-atherogenic role of the vascular endothelium and accelerate atherosclerosis. Both insulin resistance and endothelial dysfunction appear to precede the development of overt hyperglycemia in patients with type 2 diabetes. Therefore, in patients with diabetes or insulin resistance, endothelial dysfunction may be a critical early target for preventing atherosclerosis and cardiovascular disease”²

Understanding and treating endothelial dysfunction is a major focus in the prevention of vascular complications caused due to diabetes. Comparing the function of endothelial cells from normal and diabetic donors becomes crucial to better understand the deficiencies in endothelial cells from diabetic donors. Lonza’s broad offering of human endothelial cells from normal and diabetic type 1 and 2 donors is better suited to support such studies.

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Diabetes-related Differential Gene Expression in HAECs and ADSCs

With the prevalence of diabetes growing worldwide, the availability of primary human cells from diabetic donors is critical to increase research and knowledge about the disease at a cellular level. In this study, we sought to identify genes differentially regulated in diabetic type 1 and type 2 adipose-derived stem cells (ADSCs) and human aortic endothelial cells (HAECs).

Select References with Lonza’s Primary Endothelial Cells from Normal and Diabetic Donors

Down-regulation of miR-200b increases VEGF, mediating structural and functional changes in the retina in diabetes. Ruiz,et al. were trying to establish a relationship between PRC2 (Polycomb Repressive Complex 2) and microRNA miR-200b to understand the mechanisms regulating miR-200b in diabetes. Lonza’s human dermal microvascular endothelial cells (HDMECs) isolated from non-diabetic and type 1 and type 2 diabetic individuals were utilized and grown in Lonza’s Endothelial Medium. RT-PCR analysis of miR-200b, PRC2 and VEGF in HDMEC isolated from normal donors and from individuals with type 1 or type 2 diabetes helped to prove that PRC2 regulates miR-200b in retinal endothelial cells through H3K27me3 repression and was one of the first investigations to connect histone methylation and miRNA regulation in the context of diabetic retinopathy. Furthermore, the study provided evidence that restoration of miR-200b may be therapeutically beneficial.
Tsukahara, et al. were trying to understand the mechanism of the cPA-PPARγ axis in the coronary artery endothelium, especially in patients with type II diabetes. Lonza’s human coronary artery endothelial cells (HCAECs ) from normal and type 2 diabetic donors were grown in Lonza’s EGM^TM-2 Growth Medium to support the study.

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References

¹ http://circres.ahajournals.org/content/87/10/840

² https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2350146/

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