Cellular Chirality and Cellular Tissue Indexes in Vascular Morphogenesis

1. Introduction

The number of papers published involving vascular morphogenesis (which was used as a search key word) has increased exponentially. With only one article published in 1948, over the last five years, the number of documents published in PubMed exceeds 2689 ( date of accession 05.10.2023. In this review, cellular and tissue indices are discussed in the literature on vascular morphogenesis. Data regarding this review were curated using the Pubmed database. The words used to generate the data were vascular morphogenesis, cellular indices, and tissular indices.

As expected, the amount of data generated is considerable. The ultimate goal of the vascular morphogenesis study is to enable the development of organized vascular tissue that presumably might be used to replace the disease-affected one [1,2].

Growing new vessels requires a coordinated cellular response to growth. This response is sensed and triggered by cell surface receptors responsible for activating an intracellular cascade that initiates migration and controls cell growth. While the dominant molecular mechanisms have been determined, the distinct and detailed interactions remain unknown [3].

Experiments were performed using mainly mice and zebrafish models. A series of pathways have been identified. These pathways include VEGF-VEGFR, Notch-DSL, Tie-Angiopoietin, VE-cadherin and Ephrin-Eph. Other signaling pathways contributing to further vascular remodeling include plexins, TGF-beta, PDGF, and integrins [4]. In the following paragraphs, the VEGFR pathway, together with its receptors, is briefly discussed.

2. VEGFR Signaling Pathway in Vascular Morphogenesis

Coordinated cellular response is critical for the development of new blood vessels. The cellular response implies the cell surface receptors. Even if the dominant molecular motifs have been determined, how they interact is still not understood entirely [5].

The dominant pathways are the VEGF-VEGFR, Notch-DSL, Tie-Angiopoietin, VE-cadherin, and Ephrin-Eph [6,7].

The VEGF pathway is one of the critical regulators of the angiogenesis process. The VEGF/VEGF-receptor axis comprises multiple ligands and receptors with overlapping and distinct ligand-receptor binding specificities. The VEGF-receptor pathway activates signaling processes that promote endothelial cell growth, migration, and survival from preexisting vasculature. In addition, VEGF mediates vessel permeability [8].

VEGF is crucial in vasculogenesis and angiogenesis. VEGF activity is restricted mainly to vascular endothelium cells, although it does affect other cell types (e.g., stimulation monocyte/macrophage migration). In vitro, VEGF stimulates endothelial cell mitogenesis and cell migration [9].

All members of the VEGF molecular family determine a cellular response by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing a dimerization and activation through transphosphorylation [10].

Five VEGF molecules are known: VEGF A-E. VEGFA binds to VEGFR1 (Flt-1) and VEGFR -2 (Flk-1/KDR). VEGF-B stimulates VEGFR1 (Flt-1). VEGF-C interacts with VEGFR -2 (Flk-1/KDR) and VEGFR-3(Flt-4). VEGF-D stimulates VEGFR -2 (Flk-1/KDR) and VEGFR-3(Flt-4). Lastly, VEGFR-E enables VEGFR -2 (Flk-1/KDR)[11,12,13,14,15] Figure 1).

The VEGFA gene encodes VEGF-A—a platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) family member. The protein encoded is a disulfide-linked homodimer [16,17].

The Vascular Endothelial Growth Factor (VEGF) signaling pathway is essential for angiogenesis, the formation of new blood vessels from pre-existing ones. VEGF attaches to its receptors on endothelial cells, chiefly VEGFR1 (Flt-1), VEGFR2 (K.D.R./Flk-1), and VEGFR3 (Flt-4), triggering a series of internal processes that govern endothelial cell activity. The principal steps of the VEGF-VEGFR pathway are as follows: (a) The binding of VEGF to VEGFRs: VEGF-A is the primary isoform of VEGF implicated in angiogenesis. It exhibits a high affinity for both VEGFR1 and VEGFR2, triggering receptor dimerization and activation.(b) Activation of VEGFRs: Following VEGF binding, VEGFRs experience auto-phosphorylation at certain tyrosine residues in their intracellular domains, resulting in the recruitment and activation of downstream signaling molecules.Ownstream Signaling Routes: The PI3K/Akt pathway is initiated when activated VEGFRs recruit phosphoinositide 3-kinase (PI3K), resulting in the synthesis of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). This stimulates protein kinase B (Akt), which governs cell survival, proliferation, and migration. The activation of VEGFR also initiates the mitogen-activated protein kinase (MAPK) pathway, encompassing extracellular signal-regulated kinase (ERK). ERK signaling modulates endothelial cell proliferation and migration. Regulation of Gene Expression: The activation of VEGFRs and subsequent signaling pathways results in alterations in gene expression, notably the upregulation of pro-angiogenic factors such as endothelial nitric oxide synthase (eNOS) and matrix metalloproteinases (MMPs), which facilitate endothelial cell proliferation, migration, and tube formation. (e) Responses of Endothelial Cells: The signaling processes initiated by the VEGF-VEGFR connection promote endothelial cell proliferation, migration, and survival, resulting in the development of new blood vessels.Negative Regulation: The VEGF-VEGFR pathway is stringently controlled by multiple mechanisms, including the function of soluble VEGFRs (sVEGFR1 and sVEGFR2) that serve as decoy receptors, sequestering VEGF and inhibiting its interaction with membrane-bound VEGFRs. Role of Co-Receptors: Neuropilins (NRP1 and NRP2) function as co-receptors for VEGFRs and regulate VEGF signaling. They augment VEGF affinity for VEGFR2 and modulate VEGF-mediated angiogenesis [18,19]. In the following, some relevant VEGFR 1-3 are described briefly.

2.1. VEGFR1

Expression of VEGFR1 depends on the microenvironment, which is distinctively regulated under hypoxic and inflammatory conditions. VEGFR1 activation can affect vascular permeability and induce macrophage and microglia production of proinflammatory and pro-angiogenic mediators. Also, the ability of the VEGFR1 ligands (VEGF-A, PlGF, VEGF-B) to compete against each other for receptor binding and to heterodimerize is remarkable. Clinically, anti-VEGF drugs have proven effective in proliferative diseases, and their impact on the modulation of VEGFR1 signaling is still an opportunity for further research [20]. VEGFR1 may have a role as a negative regulator of embryonic angiogenesis. It promotes the PGF-mediated proliferation of endothelial cells. It has a very high affinity for VEGFA and relatively low protein kinase activity. Also, it functions as a negative regulator of VEGFA signaling. Furthermore, VEGFR1 stimulation led to phosphorylation of the regulatory subunit of phosphatidylinositol 3-kinase (PIK3R1). VEGFR1 is also implied in the activation of MAPK1/ERK2, MAPK3/ERK1, the M.A.P. kinase signaling pathway, and the AKT1 signaling pathway [21].

VEGFR1 promotes endothelial tubule branching in an organotypic angiogenesis model via a mechanism that requires Rab4A and alphavbeta3 Integrin. A recycling pathway regulated by Rab4A is an effector of VEGFR1 during the branching morphogenesis of the vasculature [22]. The VEGFR1 gene encodes a receptor tyrosine kinase and a secreted splice variant. Epidermal growth factor /fibroblast growth factor two (EGF/FGF2)-mediated VEGFR1 induction is mediated via the functional interaction of transcription factors ETS1 and hypoxia-inducible factor 2 alpha(HIF-2alpha). Mechanistic analyses revealed that EGF/FGF2 signaling induces ETS1 expression in endothelial cells, increases HIF-2alpha protein level without hypoxia, and recruits both protein C-ets-1(ETS1) and HIF-2alpha to the VEGFR1 chromatin domain [23]. VEGFA regulates embryonic angiogenesis through vascular endothelial growth factor receptor 2 (VEGFR2) expressed in the endocardium. It is shown that VEGFR1 produced in the endocardium negatively regulates embryonic coronary angiogenesis by limiting the Vegf-Notch signaling [24]. VEGFR1 and VEGFR2 expressions are upregulated during copy number variation (C.N.V.) pathogenesis. Both MF1 and DC101 significantly suppressed C.N.V. at 50 mg/kg [25]. The role of VEGF receptor 1 (VEGFR1) signaling in angiogenesis and tissue growth in an endometriosis model showed that VEGFR1 is implied in this process. VEGFR1 signaling in host-derived cells is essential for cell growth [26,27]. VEGFR1 exists in different forms, resulting from alternative splicing of the same gene. Moreover, sVEGFR-1 and sFlt1-14 are additional in angiogenesis [28]. A recycling pathway regulated by Rab4A is a critical effector of VEGFR1 during the branching morphogenesis of the vasculature [29]. Tyrosine-protein kinase acts as a cell-surface receptor for VEGFA, VEGFC, and VEGFD. The binding of vascular growth factors to isoform one leads to the activation of several signaling cascades. Activation of PLCG1 stimulates the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphateConsecutivelly they stimulate protein kinase C [30]. Angiogenesis stimulates endothelial cells (E.C.) by various cytokines and growth factors [31]. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFR-1) and K.D.R. (VEGFR-2). VEGF is essential for the early development of the vasculature [32].

The properties of VEGFR1 are summarized in Table 1:

In the following, VEGFR2, a fundamental structure in the vasculogenesis process, is discussed.

2.2. VEGFR2

VEGFR-2 is a 210-230 kDa glycoprotein expressed in vascular endothelial cells and binds VEGF-A. VEGFR-2 is closely related to VEGFR-1, for they have expected and specific ligands. Furthermore, VEGFR-2 is a highly active kinase in contrast with VEGFR1, an impaired tyrosine kinase receptor. The signaling pathways, Y1175 and Y1214, are the main autophosphorylation sites of the human VEGFR-2 when VEGF is bound [45,46,47].

VEGFR-2 is a part of the VEGF family. It is essential for developmental and reparative angiogenesis [48]. VEGF activates VEGFR2, situated in the endothelial cell membrane. VEGFR2 interacts with VRASP(), PLCγ, ScK, Cdc42, Src, and subsequently PI3K. VRAP and Sck do not engage in further interactions with other signaling molecules. PLCγ interacts with diacylglycerol and inositol trisphosphate. Cdc42 engages with p38. PI3K activates PIP3. IP3 engages with Ca+ and PKC, initiating a cascade in which SPK connects with Ras, and then engaging with Raf-1, so boosting M.E.K., which interacts with ERK. ErK enhances vascular proliferation through its interaction with DNA. Calcium ions (Ca+) are activated by inositol trisphosphate (IP3), which then engages with calnexin (CALN), facilitating the generation of nitric oxide synthase (NFTA) that interacts with cyclooxygenase-2 (COX2); the COX2 pathway subsequently triggers the synthesis of prostaglandin I2 (PGI2). FAK activation subsequently activates focal adhesion and cellular migration. Additionally, p38 activates M.A.P., thereby enhancing actin rearrangement through its interaction with HSP27, ultimately resulting in cell migration. Additionally, AktPKE activates eNos, Casp9, and Bad, which further enhances cell permeability, migration, and survival. Numerous studies indicate that VEGFR-2 is the principal mediator of VEGF-induced responses in endothelial cells. It serves as a vital signal transducer in both physiological and pathological angiogenesis [49,50].

Regarding the Notch signaling pathway, Delta notch or Seratt-like ligand, with the help of O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase (Fringe), stimulates the Notch receptor that further promotes the Notch intracellular domain. The Notch domain simulation acts on D.N.A. that additionally produces Hes 1/5(hes family bHLH transcription factor 1), Hey(hes related family bHLH transcription factor with YRPW motif 1), PreTα(pre T cell antigen receptor alpha), and NRAP( Notch-regulated ankyrin repeat-containing protein) respectively. On C.S.L. (recombination signal binding protein for immunoglobulin kappa J region like) act a series of activators -MAML, H.A.T.s, SKIP and a series of Co suppressors- SMRT, CtBP, Gro/TLE, C.I.R., SHARP, HDAC, ATXN1L, Hes1, and NRARP respectively. The Notch signaling pathway is crucial for proper embryonic development [51].

VEGF exerts biological effects through specific receptors on endothelial cell surfaces, with VEGFA/VEGFR2 signaling prominently mediating cellular responses in angiogenesis. Although weaker, VEGF/VEGFR1 signaling converges to the VEGFR2 pathway, activating multiple downstream pathways that promote endothelial cell proliferation [52]. VEGFR2-dependent activation of PI3K-AKT-mTOR signaling regulates cell survival, proliferation, anti-apoptotic functions, and cell permeability. Another crucial VEGF-mediated cell proliferation pathway involves PLCI-mediated activation of PKC, leading to downstream induction of the ERK pathway and another PKC-dependent pathway. Endothelial cell migration is influenced by VEGFA/VEGFR2 signaling through activation of p38MAPK (actin polymerization) necessary in directed migration [53].The VEGFA/VEGFR2 signaling network compiles literature data, especially regarding VEGFA-165, through VEGFR2 in endothelial cells [54]. Signaling molecules—categorized into enzymes, receptors, and transcription factors, undergo contextual activation/deactivation downstream of VEGFA/VEGFR2 signaling, influencing angiogenesis. The interactive VEGFA/VEGFR2 signaling network has a critical role in [55,56]. FAK regulates VEGFR2 and several other angiogenesis-related genes while influencing VEGFR2 and VEGF protein expression in TNBC cells [57]. Despite efforts, clinical success in promoting sprouting angiogenesis in the skeletal muscles of individuals with peripheral artery disease has not been achieved [58].

Vascular endothelial growth factor-A (VEGF-A) is essential for endothelial cell functions associated with angiogenesis. Signal transduction networks initiated by VEGFA/VEGFR2, the most prominent ligand-receptor complex in the VEGF system, lead to endothelial cell proliferation, migration, survival, and new vessel formation involved in angiogenesis [59]. Cell signaling governs cellular behavior and is subject to tight spatiotemporal regulation. Signaling output is modulated by specialized cell membranes and vesicles containing unique lipids and protein combinations. The phosphatidylinositol 4,5-bisphosphate (P.I. (4,5)P2 ), an essential component of the plasma membrane and other subcellular membranes, is involved in multiple cellular processes [60]. Rap1a and Rap1b, two highly homologous small G proteins, are required for angiogenesis in vivo and normal E.C. responses to VEGF. These results provide an insight into the role of Rap1 in VEGF signaling in E.C.s [61].

Coronary vessels in embryonic mouse hearts originate from various progenitor populations, including sinus venosus (S.V.), endocardium, and proepicardium [62]. Despite the unknown role of hypoxia and its downstream signaling in coronary vessel development, some studies have explored this aspect, identifying SOX17- and VEGF-R2-mediated signaling as potential downstream pathways influenced by hypoxia [63]. S.S.A. inhibits angiogenesis and tumor growth by blocking the VEGFR2-mediated signaling pathway [64]. In response to hypoxia, VEGF promotes angiogenesis by inducing endothelial cell sprouting, proliferation, and migration. Inducing arteriogenesis after cardiac or cerebral arterial occlusion can reduce ischemia and improve disease outcomes, with endothelial VEGF receptor 2 (VEGFR2) signaling governing both processes [65]. Identifying angiogenic factors, such as perlecan, in vertebrate development enhances the understanding of the molecular basis of angiogenesis and may inform angiogenesis-based therapeutic approaches [66].

Moreover, focal adhesion kinase (FAK) is essential in embryonic angiogenesis, governing endothelial cell (E.C.) survival and barrier functions via both kinase-independent and -dependent mechanisms. EC-specific tamoxifen-inducible FAK knockout and FAK kinase-defective (K.D.) mutant knockin mice were created to examine the role and kinase activity of FAK in adult angiogenesis. The absence of FAK or its kinase activity diminished endothelial cell proliferation and migration, suggesting that FAK predominantly functions as a kinase in the regulation of adult endothelial cell-mediated angiogenesis. Subsequent research utilizing mouse E.C. line MS1 cells demonstrated that FAK regulates VEGFR2 expression, necessitating both FAK kinase activity and its nuclear translocation.[67]. VEGF, a crucial regulator of angiogenesis, and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are currently targeted in therapeutic strategies for vascular disease. NRP1 is critical in vascular morphogenesis [68]. In the cardiovascular system, VEGF binding to VEGF receptor 2 (VEGFR-2) promotes blood vessel development [69]. However, the activated receptor signals to discrete downstream pathways, coreceptors, and distinct VEGF isoforms modulate the equilibrium between these pathways [70]. Receptor-interacting protein kinase 3 (RIPK3) is a multifunctional intracellular protein recognized as a vital component of the necroptosis-programmed cell death pathway [71]. Some VEGFR2 properties are listed in the table below (Table 2):

VEGFR3’s relevant properties are discussed in the following paragraph.

2.3. VEGFR3

Compared with VEGF-A activation of VEGFR2, VEGF-C-induced VEGFR3 activation led to a more extensive A.K.T. activation, whereas activation of ERK1/2 displayed a distinctly different kinetics [84]. Tyrosine-protein kinase acts as a cell-surface receptor for VEGFC and VEGFD. It plays an essential role in adult lymphangiogenesis and the development of the vascular network and the cardiovascular system during embryonic development. Signaling by activated FLT4 leads to enhanced production of VEGFC [85,86]. Phosphorylation in response to H2O2 is mediated by a process that requires S.R.C. and PRKCD activity. Phosphorylation at Tyr-1068 is required for autophosphorylation at additional tyrosine residues [87,88].

Elevated VEGFR2 activity in postnatal retinas following VEGFR3 deletion or VEGFR3 silencing in cultured endothelial cells reduced vascular endothelial cadherin localization at cell-cell junctions. Simultaneous deletion of VEGFR2 prevented VEGF-induced excessive vascular leakage in Vegfr3-deficient mice. VEGFR3 limits VEGFR2 expression and pathway activity, preventing excessive vascular permeability in quiescent and angiogenic blood vascular endothelial cells [89,90]. The impact of VEGFR3 on lymphatic capillary junctions remains incompletely understood, as excessive VEGFR2 signaling can remodel and seal these junctions [91]. Knockdown of FLT4 in human lymphatic endothelial cells results in impaired NOTCH1 expression and activation, with overexpression of NOTCH1 rescuing button junction formation and interstitial molecule absorption in Flt4 knockout vessels [92,93,94,95,96]. Genetic evidence suggests that VEGFR3 regulates early vessel branching and filopodia formation in the mouse brain, likely mediating the brain vascular phenotype [97,98,99]. During embryonic development, angiogenesis is initiated as mesoderm mesenchyme cells differentiate into angioblasts expressing VEGFR-2[100,101,102,103]. Hypoxia induces angiogenesis, and the injection of VEGFA enhances angiogenesis in animal models. Nonetheless, clinical trials did not reproduce the encouraging outcomes shown in animal models, perhaps due to the methods of delivery and VEGFA’s capacity to enhance arterial permeability. Recent trials validate the safety of VEGFA administration in humans, facilitating advancements in pro-angiogenic treatment strategies.[104,105,106,107,108]. Various VEGFA isoforms, including VEGFA111, VEGFA121, VEGFA145, VEGFA148, VEGFA162, and VEGFA165, are commercially available and being tested [109,110,111,112,113,114].

In the table below, some VEGFR3 properties are summarized (Table 3).

2.4. Pro-Angiogenic Therapy

Ischemic disease (heart failure, strokes, peripheral artery disease) and also some degenerative are known to be the consequence of ultimately poor blood supply. In this respect, treating these pathologies using pro-angiogenic targeting molecules is tempting. As aspected, VEGFA has the predominant role in vessel formation, growth, and branching [28,127,129,130,131]. In this respect, VEGFA is the prototype of a pro-angiogenic molecule. VEGFA acts primarily on VEGFR2. Also, due to its complex roles, VEGFR1 is stimulated by VEGFA. Furthermore, stimulation of VEGFR1 and VEGFR2 is a potential pro-angiogenic therapy [132,133]. Therapeutic angiogenesis shows excellent potential in light of the experimental results. Sadly, no F.D.A.-approved molecule exists [134]. Due to extensive research in this area, the pro-angiogenic molecules are well-categorized. Angiogenic proteins, gene therapy, peptide drugs, and organic molecules are all well-established research areas [135].

Peptides are smaller molecules( around 50 Aa) that, unlike proteins, do not need a tertiary and quaternary structure to be biologically active. Morvore Aa can be easily manipulated and optimized to match the arrangement of Aa present in angiogenesis stimulation molecules. Furthermore, the peptides can be conjugated with organic molecules, or their primary and secondary structures can be modified to fit the desired ADME properties. Given their smaller size and relatively simple structure, the pro-angiogenic peptides can be easily synthesized and released in large amounts to stimulate the angiogenesis pathway (Table 4).

Gene therapy for promoting angiogenesis involves introducing genetic material into cells to stimulate the formation of new blood vessels. This approach is explored for various medical conditions, including ischemic diseases, wound healing, and cardiovascular disorders. Here are some examples of pro-angiogenic gene therapies. Some gene therapies are listed in the table below (Table 5):

In the following paragraph, the crucial role in vascular morphogenesis of HIF 1 α Gene is presented.

2.5. HIF-1α Gene

The HIF1α gene, also known as Hypoxia Inducible Factor 1 Subunit Alpha, is crucial in cellular and systemic responses to hypoxia (low oxygen levels). It encodes the alpha subunit of the HIF-1α transcription factor, which forms a heterodimer with a beta subunit. Function: HIF-1α functions as a master regulator by activating the transcription of various genes involved in energy metabolism, angiogenesis (formation of new blood vessels), apoptosis (programmed cell death), and other genes that enhance oxygen delivery or facilitate metabolic adaptation to hypoxia [166]. Importance in Embryonic Vascularization: HIF-1 is essential for blood vessel development during embryogenesis. Tumor angiogenesis: It contributes to the growth of blood vessels in tumors; ischemic disease: HIF-1 α plays a role in the pathophysiology of conditions related to inadequate blood supply, such as ischemic heart disease. H1F-1α variants are alternatively spliced transcript variants encoding different isoforms of H1F1 α have been identified. Its main product is Hypoxia-inducible factor 1-alpha with its crystallographic structure 4h6j [167]. The Aa sequence is the following(Uniprot ID Q16665) HIF1 is a heterodimeric basic helix-loop-helix structure consisting of two subunits: HIF1A (the alpha subunit) and the aryl hydrocarbon receptor nuclear translocator (Arnt, the beta subunit). HIF1A comprises a primary helix-loop-helix domain near the C-terminal, followed by two P.A.S. (PER-ARNT-SIM) domains and a P.A.C. (PAS-associated C-terminal) domain. Additionally, the HIF1A polypeptide contains a nuclear localization signal motif, two transactivating domains (CTAD and NTAD), and an intervening inhibitory domain (I.D.) capable of repressing the transcriptional activities of CTAD and NTAD. Although three HIF1A isoforms are generated by alternative splicing, isoform1 is considered the canonical structure and has been extensively studied in terms of structure and function.

The transcription factor HIF-1 is crucial in mammalian cellular responses to systemic oxygen levels. Its activity, governed by various post-translational modifications like hydroxylation, acetylation, and phosphorylation, regulates multiple genes, including VEGF and erythropoietin. These genes are pivotal in processes such as angiogenesis and erythropoiesis, facilitating increased oxygen delivery to hypoxic areas. HIF-1 also orchestrates the transcription of genes vital for cell proliferation survival and glucose and iron metabolism. In response to fluctuating oxygen levels, HIF-1 undergoes conformational changes and binds to hypoxia-responsive elements (H.R.E.s) on gene promoters, thereby initiating transcription.

Currently, HIF-1α is shown to be implied in proteins involved in cellular responses to changes in oxygen concentration. Extended exposure to hypoxia triggers various cellular mechanisms in skeletal muscles to compensate for limited oxygen levels. One such mechanism involves upregulating specific hypoxia-inducible genes, including vascular endothelial growth factor (VEGF). The VEGF gene is crucial for angiogenesis in skeletal muscles, as evidenced by reduced capillarity upon its deletion in mice. While hypoxia-inducible factor-1alpha (HIF-1alpha) is known to activate VEGF gene transcription by binding to its hypoxic response element (H.R.E.) on the promoter, additional pathways may also contribute to VEGF upregulation during acute or prolonged hypoxia. These pathways, stimulated during hypoxic exposure, may involve inflammation, potentially related to reactive oxygen species generation or changes in cellular energy status indicated by A.M.P. kinase activity. These mechanisms offer different means of VEGF regulation in long-term hypoxic conditions such as those experienced at high altitudes. This review will delve into the cellular signals from hypoxic exposure that could enhance myocyte VEGF expression, including decreased intracellular oxygen levels, skeletal muscle inflammation involving cytokines and oxidative stress, and increased A.M.P. kinase activity and adenosine levels associated with reduced cellular energy potential [168]. Some recent findings include pancreatic cancer and hif-1α. Pancreatic cancer, a leading cause of cancer-related deaths worldwide, thrives in a highly hypoxic tumor microenvironment. HIF-1α plays a central role in the carcinogenesis and progression of pancreatic cancer. Researchers have explored how HIF-1α regulates tumorigenesis and progression in pancreatic cancer. Targeting HIF-1α and its signaling pathways could hold promise as a therapeutic approach for pancreatic cancer [169]. Other studies include retinoic acid-induced differentiation; a study investigated HIF-1α‘s role in retinoic acid-induced differentiation in SH-SY5Y cell cultures under normoxic conditions and low serum concentration. It was found that reduced serum concentrations led to cells remaining in the G0 phase, facilitating cell differentiation [170]. Also, HIF-1α is implied in adipose tissue remodeling. However, the specific mechanisms and implications of HIF-1α in this context are being studied [171]. Also, HIF-1α impacts cancer metabolism, while HIF-1α activation significantly influences cancer cell metabolism. It leads to increased glycolysis and impaired mitochondrial function in tumor cells [172]

While not directly targeting HIF-1α, gene therapy research in P.A.D. aims to improve blood flow and tissue repair. Various approaches, including growth and angiogenic factors, are being investigated in randomized controlled trials [173]. Also, HIF-1α expression and release following endothelial injury are analyzed. Researchers have quantified HIF-1α expression after administration of DMOG (a prolyl hydroxylase inhibitor) in endothelial cells. Understanding HIF-1α dynamics during endothelial injury may provide insights into vascular repair mechanisms [174].

Lastly, in a novel limb ischemia model, intramuscular delivery of AdCA5 (an adenovirus encoding a constitutively active form of HIF-1α) improved perfusion and arterial remodeling. HIF-1α regulates angiogenic growth factors, making it a potential therapeutic target for ischemic conditions. However, current studies do not demonstrate these results that explain the presumably positive benefit as a placebo effect after therapy administration [175,176,177,178].

Overall, the latest developments regarding the VEGFR pathway are summarized in the table below (Table 6):

In the next paragraph, cell chirality in the light of the vasculogenesis process is presented.

2.6. Cell Chirality Vasculogenesis

The majority of macromolecules present in cells exhibit chirality, indicating that they cannot be overlaid onto their mirror images. Nonetheless, cells can exhibit chirality, a topic that has garnered minimal attention until recently.

Consequently, chirality at the cellular level may significantly influence left-right asymmetric development in several invertebrate species. Recent reports indicate that cell chirality has been observed in numerous cultured vertebrate cells, with research suggesting that this phenomenon is evolutionarily conserved, highlighting the critical function of the actin cytoskeleton. The biological functions of cell chirality in vertebrates are still to be elucidated. However, it may regulate left-right asymmetric development or other morphogenetic processes. The exploration of cellular chirality has recently commenced, and this emerging discipline is expected to yield significant insights in biology and medicine.

The helical structure of cells is recognized for its essential function in development and illness. Nonetheless, the fundamental mechanism driving this behavior remains predominantly unexamined, especially in replicating it within rigorously regulated engineering systems. Utilizing sophisticated microfluidics it provided substantial evidence of the spontaneous formation of helical endothelial tubes demonstrating strong right-handedness dictated by intrinsic cell chirality. Modulating endothelial cell chirality with small-molecule pharmaceuticals induces a dose-dependent inversion of handedness in constructed arteries alongside non-monotonic alterations in vascular permeability.

The morphogenesis of tubular tissues or organs is a crucial process that takes place during early development in various animals. Structures like the embryonic heart tubes in vertebrates and the embryonic hindgut in Drosophila are among the initial instances of left-right (LR) symmetry breakdown. Prior research has shown that left-right asymmetry during tubular morphogenesis might arise from cellular chirality. The inherent left-right asymmetric feature of cells is involved in diverse processes across different morphological scales, with the cellular chiral bias showing significant alignment with the handedness observed at the multicellular or tissue level. Despite significant advancements in elucidating the significance of cell chirality in the tubular morphogenesis of epithelial and cardiac tissues, the understanding of left-right asymmetry in typical endothelial blood vessels, another common tubular form, remains considerably constrained.

Furthermore, in the natural environment, blood arteries are predominantly lined with endothelial cells on the inner surface of the cylinder wall, with their apical side oriented towards the vessel’s center. These cells provide a compact endothelial lumen, delineating the intravascular milieu from the extravascular space and facilitating the dynamic modulation of vascular permeability. It was previously observed that endothelial cells have a pronounced clockwise (CW) chiral bias. The neutralization or randomization of this CW bias led to junctional disruptions and increased endothelial permeability due to the failure to establish intact connections between cells exhibiting heterogeneous chirality. Although the regulatory roles of cell chirality in endothelial functions are well-established, contemporary research on the morphological and biophysical characteristics of tubular vessels has predominantly concentrated on cell behaviors or cues that are longitudinal, circumferential, or perpendicular to the curved substrate surface, neglecting the implications of cell handedness or related biases in cell alignments and mechanical forces.

Also, the demonstration of LR asymmetry or chirality in a system generally necessitates two established axes, namely the apical-basal (AB) axis and the front-rear (FR) axis. Nonetheless, the polarization of the tissue along the longitudinal axis is not consistently evident, as observed in a growing heart when no directional guidance is present. Blood flow is directed. It is missing during the earliest phase of vascular formation and angiogenesis, therefore failing to elucidate cell polarization completely. This prompts an inquiry about the manifestation of the chiral characteristic of endothelial cells morphologically on the tubular substrate, given the presence of just the AB axis. While blood arteries do not experience tissue or organ-scale asymmetrical morphogenesis akin to the chiral C-looping of heart tubes, numerous investigations utilizing in vitro three-dimensional (3D) vascular platforms demonstrate a helical alignment of endothelial cells along tubular geometries. This issue has not been thoroughly examined, and handedness has not been objectively evaluated.

Endothelial cells can autonomously develop a right-handed helical structure in both in vitro tubular substrates and in vivo vascular tissues. The reversal of inherent cellular chirality modified the helical handedness of the vasculature, corroborated by in vitro tests, including PKC activation and computer simulations of chiral torque force direction switching.

In vitro models have been extensively utilized in biomedical research. Over the past century, Petri plates have served as a fundamental model for cell culture, yielding significant insights into the biophysical and metabolic characteristics of cells within live organisms. Transwell membranes facilitate epithelial cell culture by establishing the appropriate apicobasal polarity observed in vivo, marked by pronounced actin and atypical protein kinase C expression at the apical surface, with the deposition of basement extracellular matrix at the basal surface. The in vitro scratch experiment, which involves creating a linear gap in a cellular monolayer, facilitates the investigation of cell migration during wound healing and the analysis of front-rear polarity. These approaches have facilitated considerable research on apicobasal and front-rear polarity in many pieces of literature, resulting in a thorough grasp of molecular pathways thanks to these straightforward in vitro models. The polarity along the left-right (LR) axis has not been extensively investigated at the cellular level in a controlled manner until the recent emergence of various in vitro cell chirality systems.

The formation of the LR axis is essential for living creatures. All vertebrates demonstrate asymmetry along the body’s midline, referred to as handedness or chirality, in the arrangement and structure of internal organs. A variation from this arrangement frequently results in significant implications, particularly when one or two organs are positioned in a mirrored orientation (i.e., situs ambiguus). In cases of situs inversus, where all internal organs are transposed to the opposite side of the body, individuals may remain healthy; nevertheless, some may experience conditions such as Kartagener syndrome. Various models have been developed to elucidate the mechanisms of symmetry breaking in animal embryos, focusing on critical factors such as the leftward fluid flow at the ventral node induced by ciliary rotation, voltage gradients arising from the asymmetric expression of ion channels, and asymmetric vesicular transport mediated by unconventional myosin ID along actin cable network. Laterality defects are present in more than 0.1% of live births. The actual percentage may be higher, as inadequate axis establishment in fetuses frequently results in miscarriages. Consequently, investigating the establishment of the LR axis holds considerable clinical significance.

Growing data indicates that cell chirality may play a crucial role in the left-right asymmetry of embryonic development. An object (e.g., D-glucose and DNA) is deemed chiral when it can be differentiated from its mirror image. Cell chirality mathematically refers to the relationship of handedness (left or right) between the LR axis and the established apicobasal axis (often arising from two-dimensional cell attachment) and the front-rear axis (generally determined by the nuclear-centrosomal axis in polarized cells). Chirality can also be recognized as the directional rotation of cellular organelles, the cytoskeleton, and whole cells. Due to the inherent randomness in the morphology and movement of individual cells, cell chirality is typically characterized as a statistical property of populations at subcellular, cellular, and multicellular levels. It is quantified through the directional biases in organelle positioning, cytoskeletal dynamics, cell shape, alignment, and migration. Cell chirality has been documented in various biological systems throughout developmental biology. Xenopus embryos and parthenogenetically activated eggs, when cleaved and treated with the myosin ATPase inhibitor 2,3-butanedione monoxime, demonstrated significant chiral twisting of actin structures. The chirality of planar cell shape was identified as the determinant of directional looping in the hindgut of Drosophila. Comparable outcomes were seen for the rotation of Drosophila genitalia. Formin, an essential scaffolding protein linked to cellular chirality, was identified as being associated with left-right asymmetry in the pond snail and frog.

Research on LR asymmetry has been gained from in vitro models of cellular chirality. Initially, in vitro models integrate minimal stimuli by omitting potential confounding variables, hence significantly enhancing the elucidation of molecular pathways. Secondly, akin to Petri plates, in vitro systems are generally user-friendly, allowing for reliable imaging and molecular tests. Third, these approaches may be more effective in screening teratogens and environmental variables linked to laterality-related birth abnormalities. Ultimately, utilizing human cells, in vitro investigations may elucidate patient-specific pathways linked to deformities (Table 7).

The cellular chirality indexes that play a role in vasculum genesis are discussed in the following paragraph.

2.7. Cellular Chirality Indices

Cellular chirality indices are quantitative metrics employed to characterize and quantify the chirality (or handedness) in the structure or activity of cells. Cellular chirality is a characteristic observed in multiple biological situations, ranging from tissue-level asymmetries to the morphologies and behaviors of individual cells, including cell migration. Measuring chirality aids researchers in comprehending the emergence of left-right asymmetry in biology, influencing areas such as developmental biology, tissue engineering, and disease modeling (Table 8).

Furthermore, a generalized view is required, so in the next paragraph, the tissue chirality indices are presented.

2.8. Tissue Indices

Tissue indices are quantitative metrics employed to assess the organization, structure, and functionality of cellular populations within tissues. In contrast to cellular indices, which emphasize individual cells, tissue indices reflect traits that arise from the aggregate behavior and organization of several cells. These indices assist researchers in comprehending the collaborative functioning of cells to create cohesive, functional structures like organs, and they are frequently employed in investigations of tissue formation, morphogenesis, wound healing, and disease progression (Table 9).

Furthermore, the differences between cellular and tissue indices are discussed.

2.9. Cellular Indices vs. Tissue Indices

Cellular indices quantify specific features, structures, or behaviors within individual cells. They focus on parameters like cell shape, polarity, migration patterns, organelle positioning, and molecular organization within single cells. These indices help researchers study cell-level processes like cellular chirality, migration, polarity, and the effects of intracellular components on overall cell function. Cellular indices are commonly used in research on cancer metastasis, immune response, and cell differentiation. Nuclear Rotational Chirality Index, Actin Polymerization Rate Index, and Golgi Apparatus Orientation Index, all of which capture unique aspects of cellular orientation and asymmetry relevant to single-cell behaviors.

Tissular indices measure properties that emerge from the collective organization, interaction, and alignment of multiple cells within a tissue. These indices capture the behavior of cells as a cohesive unit within a tissue matrix, focusing on the structural and functional arrangement that arises when cells work together. Tissular indices are important for understanding processes like tissue morphogenesis, organ development, and wound healing, where the behavior of cell groups (rather than isolated cells) drives biological outcomes. They are also valuable in studying tissue-level chirality, such as the rotation of cell layers in developing organs. Epithelial Sheet Rotation Index, which tracks coordinated rotation in epithelial cells; Multi-cellular Swirling Chirality, used to study how groups of cells orient themselves collectively on substrates; and Tissue Polarity Index, which assesses overall alignment within tissue sections.

Cellular indices focus on intracellular and single-cell processes, while tissular indices evaluate intercellular and collective tissue dynamics. Cellular indices assess local factors (within or affecting one cell), whereas tissular indices evaluate the organization and behavior of cell populations, capturing emergent properties that are not observable at the single-cell level. Cellular indices are more relevant in studies involving isolated cells in culture, whereas tissular indices are essential in vivo studies of organ development and functional tissue organization.

Together, these two categories of indices provide a full spectrum for understanding how both individual cell properties and collective cell behaviors contribute to the structural and functional organization of tissues and organs (Table 10).

The VEGF-VEGFR pathway and implications in cellular chirality are further discussed.

2.10. VEGF -VEGFR Signaling and Cellular Chirality

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) play critical roles in cellular processes like angiogenesis (formation of new blood vessels), which is essential for supplying tissues with oxygen and nutrients. Research is increasingly revealing connections between VEGF/VEGFR signaling and cellular chirality, particularly in the context of cell migration, tissue organization, and development (Table 11).

These factors—cytoskeletal dynamics, VEGF gradients, planar cell polarity, integrin interactions, and mechanical forces—work alongside VEGF/VEGFR signaling to enhance cellular chirality. They promote directional migration, cell polarity, and organization within tissues, which are critical for developmental processes and tissue remodeling. By understanding how these pathways interact, researchers can better understand and potentially manipulate cellular behavior in contexts like angiogenesis, wound healing, and tissue engineering.

Furthermore, angiogenesis shaping methodologies using cellular /tissular indices are presented.

2.11. Angiogenesis shaping using cellular /tissular indices.

Shaping angiogenesis through cellular and tissue indices involves understanding and manipulating various factors that influence blood vessel formation. In the following table, the following methodologies are summarized (Table 12).

Gerhardt, H., & Betsholtz, C. discuss endothelial tip cell migration and the role of cellular polarity in guiding angiogenic sprouting. It highlights cellular indices like directional migration and polarity vector analysis to examine tip cell dynamics in response to VEGF gradients. This article emphasizes the role of Directional Migration and Tip Cell Selection in Angiogenesis [308].

Vion, A.C. et al. analyze how primary cilia and VEGF interact in endothelial cells to drive chiral organization and vascular patterning. Using cellular orientation indices, it evaluates how endothelial cells align and migrate to shape vessel architecture. The role of Role of Cellular Alignment and Collective Migration in Vessel Formation is summarised in this article [309]

Chen, K.D. et al. explore how mechanical cues like shear stress influence VEGF-driven angiogenesis, focusing on endothelial cell alignment and chirality under flow conditions. The study uses alignment and orientation indices to quantify cellular response to shear forces, showing how these factors contribute to angiogenic vessel shaping, emphasizing the importance of Mechanical Forces and Shear Stress on Endothelial Chirality [310]

Lamalice, L., Le Boeuf, F., & Huot emphasize the role of actin dynamics and focal adhesion distribution in endothelial cell migration. Cellular indices, such as focal adhesion rotation and actin alignment, are used to study the orientation and migration of cells during blood vessel sprouting [311].

Carmeliet, P., in his review, covers how cellular morphology and polarity affect angiogenic sprout formation. It discusses the importance of cellular shape indices in assessing how cell elongation and polarity drive structured angiogenesis [312].

Hynes, R.O. shows how Integrins, in coordination with VEGFR, play a critical role in endothelial cell adhesion, alignment, and polarity during angiogenesis. This study details how integrin-VEGFR crosstalk is assessed using indices for cell polarity and alignment to understand vascular organization [313].

Angelini, T.E. et al. describe Collective Cell Behavior and Multi-cellular Swirling in Vascular Patterning. This paper investigates collective cell migration and multi-cellular swirling patterns, applying cellular indices to measure alignment and swirling chirality. These indices are critical for analyzing coordinated cell behavior that shapes organized vascular structures [314].

Avraamides, C.J., Garmy-Susini, B., & Varner, J.A., in their review, discusses how integrin-mediated adhesion to the extracellular matrix (ECM) modulates endothelial cell orientation and chiral migration. Using cellular indices like focal adhesion density and polarity indices, this study examines how integrin signaling reinforces cellular alignment and organizes vessel structures [315].

Phng, L.-K., & Gerhardt, H. explore how Notch and VEGF signaling coordinate endothelial tip cell selection and migration. Polarity and directional indices are used to quantify cell responses in a gradient, highlighting the importance of VEGF and Notch interactions in shaping chiral migration patterns in angiogenesis [316]

Etienne-Manneville, S., & Hall investigate how Rho GTPases, downstream of VEGFR activation, influence cell polarity, migration, and actin dynamics. Cellular indices related to cytoskeletal alignment and polarity are used to study the Rho-mediated directional movement of endothelial cells in forming vascular structures [317].

Levesque, M.J., & Nerem, R.M show that this classic study examines the effects of mechanical stress, particularly shear stress, on VEGF-stimulated endothelial cell alignment and collective orientation. Using cellular orientation and alignment indices, it quantifies endothelial cell behavior under fluidic conditions, which are essential for vascular patterning [318].

Nelson, C.M., & Bissell discuss the role of cell shape and curvature in angiogenesis and how the extracellular environment influences endothelial cell organization. Cell shape indices, along with polarity indices, are used to analyze how membrane curvature guides cell-cell interactions in sprouting angiogenesis [319].

Siekmann, A.F., & Lawson, N.Dexamines how Notch signaling through lateral inhibition affects cell alignment and tip-stalk cell dynamics in angiogenesis. By applying cellular indices like alignment and polarity indices, the researchers analyze the spatial organization of cells in a vessel sprout [320].

Bentley, K. et al. show how VE-cadherin dynamics impact endothelial cell rearrangements and chiral orientation during angiogenic sprouting. The study uses chirality and alignment indices to quantify how endothelial cells align and shift to form structured blood vessels [321].

3. Conclusions

The vascular angiogenesis and subsequent morphogenesis process indicate a complicated molecular network. Additional research is required to delineate the relationships among the implicated compounds accurately. Concerning the VEGFR pathway, VEGFR2 appears to be a pivotal component. The principal components of the VEGFR pathway are the VEGF receptors VEGFR1, VEGFR2, and VEGFR3. Multiple variants of VEGF are under investigation for potential medicinal use. Investigating the NOTCH and VEGFR signaling pathways may enable the modulation, enhancement, or suppression of vascular morphogenesis.

Cellular chirality—the inherent “handedness” of cells in movement, structure, and organization—plays a pivotal role in angiogenesis, the process by which new blood vessels form from pre-existing ones. This chiral behavior is fundamental to the directional and patterned organization of endothelial cells during vascular development and remodeling. In angiogenesis, cellular chirality guides endothelial cells to adopt precise orientations and migration patterns, which are essential for creating functionally structured blood vessels that can supply tissues with the necessary nutrients and oxygen. Cellular chirality in this context is influenced by various factors, including VEGF/VEGFR signaling, mechanical forces, interactions with the extracellular matrix (ECM), and cytoskeletal dynamics.

Quantifying these complex behaviors requires a range of cellular and tissue indices that capture both individual cell characteristics and collective tissue-level patterns. Cellular indices, such as cell orientation, polarity, and migration direction, allow researchers to measure chiral behaviors at the single-cell level, providing insights into how individual endothelial cells align, polarize, and move directionally in response to angiogenic cues. Tissue-level indices, such as multi-cellular alignment, rotational chirality, and ECM alignment, complement these indices. These reveal emergent patterns as cells interact and self-organize into structured vascular networks.

Through this combined approach, cellular and tissue indices have proven essential in advancing our understanding of angiogenesis, from molecular signaling to multicellular dynamics. By systematically quantifying cell and tissue behavior, these indices enable a detailed exploration of the mechanisms by which cellular chirality shapes vascular development, guiding efforts in regenerative medicine, cancer research, and developmental biology. The integration of cellular and tissue indices thus provides a comprehensive framework for analyzing both individual and collective behaviors, ultimately illuminating the principles governing tissue organization and vascular health.