*4.4.1. Development of mammary blood vasculature*

Vasculature in the mammary gland undergoes repeated cycles of expansion and regression concomitantly with the cycles of growth, differentiation, and regression of the mammary epithelium [80]. The development of blood vessels occurs in parallel with mammogenesis. In the course of vascularization, first the process of de novo blood vessel formation takes place in the embryonic life, followed by angiogenesis which serves to form new vessels from pre-existing ones [80]. Angiogenesis is driven in main part by epithelial and stromal cells through secretion of the vascular endothelial growth factor (VEGF) and matrix metalloproteinases, especially MMP-9. Furthermore, studies have shown that development of the vessels in the mammary gland is driven by the same hormones that stimulate growth of the glandular parenchyma, that is the metabolic and sex hormones and the growth factors [81].

Before pregnancy, the mammary vasculature is composed of a thin layer of simple squamous endothelial cells forming a complex vascular network along with myoepithelial cells and connective tissue [82]. The structure of the glandular vasculature has been the best characterized in the mouse mammary gland. It is described as the basket-like capillary beds surrounding the alveoli clusters [83]. The capillary vessels run in parallel or encircle the mammary parenchyma and branch throughout the adipose tissue [82]. In humans, a high number of small capillaries are surrounding the ductal structures, whereas the acini of the lobular structures are interspersed by fewer, but significantly larger capillaries, which are sinusoidal in shape [80]. Such morphology provides a slower blood flow, thus a prolonged contact of the lobuloalveolar epithelium with circulating hormones and nutrients. During pregnancy, the growth of the mammary vessels intensifies along with expanded development of the parenchyma in order to increase the cell number and surface area to provide a maximal interface for nutrient transfer and milk secretion after parturition. Furthermore, increased surface area of the luminal endothelium is also accomplished by formation of microvilli and marginal folds on individual endothelial cells [82]. Studies on bovine model of mammogenesis showed that the blood volume expands in the pregnant animal, and about 15% of the cardiac output is directed to the fetoplacental unit toward the end of pregnancy, but at parturition most of the blood flow is redirected from the uterus to the mammary glands [81].

exposure. This function is especially relevant in regard to bovine mammary gland which is highly prone to infections due to extended period of lactation connected with intensive milk production. Exposure to pathogens initially triggers a response from MECs and resident immune cells which produce and secrete a variety of inflammatory mediators, such as cytokines. These inflammatory mediators also activate the endothelial cells, increasing vascular permeability which is necessary for the influx of neutrophils to ingest pathogens and limit extravascular tissue damage [82]. Endothelial cells produce a variety of vasoactive mediators,

inflammation, endothelial nitric oxide synthase (eNOS) becomes activated by increased intracellular calcium levels, leading to conversion of arginine to citrulline and NO. Subsequently, NO activates cellular pathways that result in inhibition of calcium influx into the endothelial cells, thus relaxation of the actin cytoskeleton. In addition to NO biosynthesis, constitutive cyclooxygenase-1 (COX-1) is activated by increased intracellular calcium and facilitates

modulate the vascular tone in order to provide an optimal endothelial surface to facilitate rolling, attachment, and migration of leukocytes that serve to regulate an appropriate immune response to infection [82]. However, during very early stages of infection and inflammation, an opposing process of vasoconstriction is also very important to protect the host's organism in the event of mechanical injury and bleeding. Interestingly, production of vasoconstrictors, such as platelet-activating factor (PAF), by endothelial cells may in turn induce increased production of NO, to prevent sustained vasoconstriction [88]. This suggests that modulation of vascular tone during the initial inflammatory response is tightly regulated to prevent

Endothelial cells, lining the extensive vascular network of the mammary gland, may also contribute to the production of inflammatory mediators, especially IL-1, IL-6, IL-8, and granulocyte colony-stimulating factor (GM-CSF), during inflammation of the mammary gland (mastitis). IL-8 directly stimulates bovine neutrophil migration, phagocytosis, priming, and enzyme degranulation. Both epithelial and endothelial cells contribute to the production of IL-8 during *Escherichia coli* infection. In cows experimentally infected with *E. coli* via injection in the teat canal, MECs showed increased levels of IL-8 mRNA until 24 h post infection, whereas endothelial cells showed increased levels of IL-8 mRNA 24 h after infection, resulting in sustained IL-8 level in tissue [89]. Studies on bovine mammary endothelial cells demonstrated that in early reaction to *E. coli* infection vascular-derived PAF seems to play a prominent role [90]. PAF is a potent phospholipid mediator and endothelial cells work as a target and a source of this molecule. In bovine mammary endothelial cells stimulated in vitro with endotoxin obtained from *E. coli,* PAF biosynthesis began as early as 30 min after the endotoxin challenge and peaked at 1 h following the challenge. The biosynthesis of PAF preceded the endotoxininduced IL-1β and IL-8 mRNA expression that reached peak expression between 4 and 12 h following stimulation. These results suggest that vascular-derived PAF is an early proinflammatory mediator during pathogen invasion in bovine mammary gland [90]. Therefore, the endothelium enables the progression of a self-limiting inflammatory response to milkproducing tissue through modulation of vascular tone and blood fluidity, vascular perme-

and oxylipid. By releasing the vasoactive mediators, endothelial cells

), endothelin-1, and histamine. At the onset of

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such as nitric oxide (NO), prostacyclin (PGI2

unnecessary damage to blood vessels and interstitial tissue [82].

ability, endothelial adhesiveness, and production of inflammatory mediators.

the synthesis of PGI2

Functional differentiation of the mammary gland during lactogenesis is also tightly connected with further changes in morphology and properties of the endothelial cells, which occur in order to support the efficient milk synthesis and secretion. The vasculature of the lactating gland is composed of a well-developed capillary meshwork enveloping the secretory alveoli with basket-like honeycomb structures [84]. The mammary endothelial cells show elevated number of mitochondria supporting their increasing demands for energy during milk production period. A higher number of pinocytotic vesicles is also observed in the endothelial cells, providing efficient transportation of plasma solutes and molecules, such as glucose [85]. In addition, increased capillary permeability occurs during early lactation. Capillaries have thinner walls and are in closer contact with the mammary alveoli, which also aids the enhanced transfer of nutrients and fluids in the functionally active gland [80, 82]. Studies done on rodents have shown that the development of the mammary vasculature, measured as the number of capillaries per individual lobular ductile, surpasses the development of the parenchymal network during lactation [82, 86]. This underlines the important role of the glandular vascular system supporting the optimal function of the mammary gland during the milk production period.

After weaning or termination of milking, when mammary gland involution takes place, the endothelium undergoes regression similarly to the mammary epithelium. Although the mechanisms controlling endothelial regression have not been well recognized so far, it seems that apoptotic cell death at least partially accounts for the remodeling of the vasculature [84]. It is worth noting that the timing of endothelial and epithelial regression is not equal, and MECs apoptosis precedes the death of the endothelial cells [84]. This indicates that the changes in the structure of the mammary gland are initiated in the parenchymal compartment and the altered microenvironment of the gland induces the changes in the vasculature. It is possible that the vascular regression is induced mechanically by disruption of the contact and anchoring between the endothelium and the collapsing mammary epithelial cells. The signals could be mediated by integrins and their cognate intracellular signal transducers, such as members of the Src family and the focal adhesion kinase (FAK); however, further studies are needed to confirm this hypothesis. Djonov and co-workers [84] also suggested that the massive endothelial regression cannot be exclusively due to apoptotic cell death since apoptotic endothelial cells were observed only occasionally in the involuting gland [87]. The authors proposed another mechanism involving regressive remodeling of the endothelium, which they termed angiomeiosis, taken from the Greek words angio (vessel) and meiosis (dwindling, retraction).

#### *4.4.2. Function of endothelial cells in immune response to infections in the mammary gland*

One of the most important functions of the endothelial cells is the ability of these cells to regulate the immune response of the host to protect the mammary gland during pathogen exposure. This function is especially relevant in regard to bovine mammary gland which is highly prone to infections due to extended period of lactation connected with intensive milk production. Exposure to pathogens initially triggers a response from MECs and resident immune cells which produce and secrete a variety of inflammatory mediators, such as cytokines. These inflammatory mediators also activate the endothelial cells, increasing vascular permeability which is necessary for the influx of neutrophils to ingest pathogens and limit extravascular tissue damage [82]. Endothelial cells produce a variety of vasoactive mediators, such as nitric oxide (NO), prostacyclin (PGI2 ), endothelin-1, and histamine. At the onset of inflammation, endothelial nitric oxide synthase (eNOS) becomes activated by increased intracellular calcium levels, leading to conversion of arginine to citrulline and NO. Subsequently, NO activates cellular pathways that result in inhibition of calcium influx into the endothelial cells, thus relaxation of the actin cytoskeleton. In addition to NO biosynthesis, constitutive cyclooxygenase-1 (COX-1) is activated by increased intracellular calcium and facilitates the synthesis of PGI2 and oxylipid. By releasing the vasoactive mediators, endothelial cells modulate the vascular tone in order to provide an optimal endothelial surface to facilitate rolling, attachment, and migration of leukocytes that serve to regulate an appropriate immune response to infection [82]. However, during very early stages of infection and inflammation, an opposing process of vasoconstriction is also very important to protect the host's organism in the event of mechanical injury and bleeding. Interestingly, production of vasoconstrictors, such as platelet-activating factor (PAF), by endothelial cells may in turn induce increased production of NO, to prevent sustained vasoconstriction [88]. This suggests that modulation of vascular tone during the initial inflammatory response is tightly regulated to prevent unnecessary damage to blood vessels and interstitial tissue [82].

on individual endothelial cells [82]. Studies on bovine model of mammogenesis showed that the blood volume expands in the pregnant animal, and about 15% of the cardiac output is directed to the fetoplacental unit toward the end of pregnancy, but at parturition most of the

Functional differentiation of the mammary gland during lactogenesis is also tightly connected with further changes in morphology and properties of the endothelial cells, which occur in order to support the efficient milk synthesis and secretion. The vasculature of the lactating gland is composed of a well-developed capillary meshwork enveloping the secretory alveoli with basket-like honeycomb structures [84]. The mammary endothelial cells show elevated number of mitochondria supporting their increasing demands for energy during milk production period. A higher number of pinocytotic vesicles is also observed in the endothelial cells, providing efficient transportation of plasma solutes and molecules, such as glucose [85]. In addition, increased capillary permeability occurs during early lactation. Capillaries have thinner walls and are in closer contact with the mammary alveoli, which also aids the enhanced transfer of nutrients and fluids in the functionally active gland [80, 82]. Studies done on rodents have shown that the development of the mammary vasculature, measured as the number of capillaries per individual lobular ductile, surpasses the development of the parenchymal network during lactation [82, 86]. This underlines the important role of the glandular vascular system supporting the optimal function of the mammary gland during the

After weaning or termination of milking, when mammary gland involution takes place, the endothelium undergoes regression similarly to the mammary epithelium. Although the mechanisms controlling endothelial regression have not been well recognized so far, it seems that apoptotic cell death at least partially accounts for the remodeling of the vasculature [84]. It is worth noting that the timing of endothelial and epithelial regression is not equal, and MECs apoptosis precedes the death of the endothelial cells [84]. This indicates that the changes in the structure of the mammary gland are initiated in the parenchymal compartment and the altered microenvironment of the gland induces the changes in the vasculature. It is possible that the vascular regression is induced mechanically by disruption of the contact and anchoring between the endothelium and the collapsing mammary epithelial cells. The signals could be mediated by integrins and their cognate intracellular signal transducers, such as members of the Src family and the focal adhesion kinase (FAK); however, further studies are needed to confirm this hypothesis. Djonov and co-workers [84] also suggested that the massive endothelial regression cannot be exclusively due to apoptotic cell death since apoptotic endothelial cells were observed only occasionally in the involuting gland [87]. The authors proposed another mechanism involving regressive remodeling of the endothelium, which they termed angiomeiosis, taken from the Greek words angio (vessel) and meiosis

*4.4.2. Function of endothelial cells in immune response to infections in the mammary gland*

One of the most important functions of the endothelial cells is the ability of these cells to regulate the immune response of the host to protect the mammary gland during pathogen

blood flow is redirected from the uterus to the mammary glands [81].

106 Stromal Cells - Structure, Function, and Therapeutic Implications

milk production period.

(dwindling, retraction).

Endothelial cells, lining the extensive vascular network of the mammary gland, may also contribute to the production of inflammatory mediators, especially IL-1, IL-6, IL-8, and granulocyte colony-stimulating factor (GM-CSF), during inflammation of the mammary gland (mastitis). IL-8 directly stimulates bovine neutrophil migration, phagocytosis, priming, and enzyme degranulation. Both epithelial and endothelial cells contribute to the production of IL-8 during *Escherichia coli* infection. In cows experimentally infected with *E. coli* via injection in the teat canal, MECs showed increased levels of IL-8 mRNA until 24 h post infection, whereas endothelial cells showed increased levels of IL-8 mRNA 24 h after infection, resulting in sustained IL-8 level in tissue [89]. Studies on bovine mammary endothelial cells demonstrated that in early reaction to *E. coli* infection vascular-derived PAF seems to play a prominent role [90]. PAF is a potent phospholipid mediator and endothelial cells work as a target and a source of this molecule. In bovine mammary endothelial cells stimulated in vitro with endotoxin obtained from *E. coli,* PAF biosynthesis began as early as 30 min after the endotoxin challenge and peaked at 1 h following the challenge. The biosynthesis of PAF preceded the endotoxininduced IL-1β and IL-8 mRNA expression that reached peak expression between 4 and 12 h following stimulation. These results suggest that vascular-derived PAF is an early proinflammatory mediator during pathogen invasion in bovine mammary gland [90]. Therefore, the endothelium enables the progression of a self-limiting inflammatory response to milkproducing tissue through modulation of vascular tone and blood fluidity, vascular permeability, endothelial adhesiveness, and production of inflammatory mediators.

#### *4.4.3. Lymphatic vasculature in the mammary gland*

When describing the vasculature present within the structure of the mammary gland, one needs to mention also the lymphatic vasculature, which plays a distinct role in the gland's function. Lymphatic vessels serve to return the interstitial protein-rich fluid to the bloodstream, absorb dietary fats and fat-soluble vitamins from the digestive tract, and traffic the immune cells to the site of their physiological destination, as well as at the time of infection [91]. Very little is known about the course of lymphatic vessel formation during mammogenesis. Betterman and co-workers described the process of lymphangiogenesis during the postnatal development of the mouse mammary gland [91]. The authors showed that lymphatic vessels share an intimate spatial association with epithelial ducts and large blood vessels. Lymphatic vessels were observed to encircle epithelial ducts in the mammary glands of virgin and pregnant mice; however, these vessels were not dispersed throughout the stroma and were excluded from alveoli during pregnancy [91]. In contrast, lymphatic vessels in the rat mammary gland were found throughout the interlobular connective tissue and in close association with the alveoli during pregnancy, pointing at substantial interspecies differences [92]. The results of the study performed by Betterman and coworkers [91] have indicated that myoepithelial cells are the source of prolymphangiogenic growth factors, such as VEGF-C and VEGF-D, that drive the expansion of lymphatic vasculature. Interestingly, the lymphatic vessels were not observed in close proximity to alveoli in the pregnant and lactating murine mammary glands. This phenomenon could be caused by insufficient prolymphangiogenic stimuli originating from myoepithelial cells which form a discontinuous sheath around the secretory MECs of the alveoli. Alternatively, the absence of lymphatic vessels could result from repulsive bioactive compounds secreted by the alveolar epithelium [91]. Among the considered molecules showing possible properties of repelling the lymphatic vascular growth is soluble VEGF receptor 2 (sVEGFR-2), which was shown to maintain the lymphatic state of cornea by sequestering endogenous VEGF-C [93].

**Acknowledgements**

05-1/KNOW2/2015.

**Nomenclature**

**Conflict of interest**

The authors declare no conflict of interest.

BM basement membrane

ECM extracellular matrix

CAFs cancer-associated fibroblasts

CSF1 colony stimulating factor-1

EGF epidermal growth factor

ERα estrogen receptor alpha FGF fibroblast growth factor

GHR growth hormone receptor

HGF hepatocytes growth factor

IGF-I insulin-like growth factor-I

MECs mammary epithelial cells

GH growth hormone

IL interleukins

EMT epithelial-to-mesenchymal transition

ERBB1 epidermal growth factor receptor

FGFRs fibroblast growth factor receptors

This work was supported by a grant no: KNOW2015/CB/PRO1/21 from KNOW (Leading National Research Centre) Scientific Consortium "Healthy Animal—Safe Food" (decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015). Publication of this chapter was funded by KNOW (Leading National Research Centre) Scientific Consortium "Healthy Animal—Safe Food", decision of Ministry of Science and Higher Education No.

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#### *4.4.4. Summary*

Mammary vasculature supports three aspects of mammary gland physiology: (1) capillary endothelial cells form a semipermeable barrier that facilitates the exchange of serum compounds to provide oxygen, remove CO<sup>2</sup> , and transfer solutes and macromolecules for cellular energy metabolism; (2) vascular endothelium provides a high rate of transfer of blood-derived components, such as glucose and amino acids for efficient synthesis of milk; (3) it also plays a significant role in orchestrating host defense to infectious pathogens, which is especially important in extensively active bovine mammary gland producing milk volumes that exceed the nutritional requirements of the offspring. Still, the intricacy of the epithelial-endothelial interactions and their impact on mammary gland development remain largely undiscovered. Further research is needed to gain more knowledge about the role of endothelial cells in the complex interactions between the stromal and epithelial compartments of the mammary gland (**Figure 2**).
